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Cell Structure & Function Antibodies and Reagents

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Table of ContentsIntroduction ....................................................................................................................................................................................3

Cell Biology Antibody Validation .............................................................................................................................................4

Cell Structure/ Organelles ..........................................................................................................................................................8

Cell Development and Differentiation ................................................................................................................................10

Growth Factors and Receptors ...............................................................................................................................................12

Cell Proliferation, Growth, and Viability...............................................................................................................................14

Cell Cycle ........................................................................................................................................................................................16

Cell Signaling ................................................................................................................................................................................18

Epigenetics and Transcriptional Regulators ......................................................................................................................20

Cell Adhesion and Extracellular Matrix ................................................................................................................................22

Cell Death .......................................................................................................................................................................................24

For Research Purpose Only. Not for use in diagnostic or therapeutic procedures. Alexa Fluor® is a registered trademark of Life Technologies.Brilliant Violet™ is a trademark of Sirigen.CyTOF® is a registered trademark of Fluidigm Corporation. DRAQ™, DyLight™, and SYTOX™ are trademarks of Thermo Fisher Scientific Inc.

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IntroductionBioLegend’s growing portfolio of reagents for Cell Biology research now includes over 700 target proteins. Our reagents can be used to detect cellular structure proteins such as nuclei and golgi, and cell function proteins for processes such as cell proliferation, cell cycle, cell signaling, and epigenetics. These reagents can be used for many different types of assays such as western blot, microscopy, immunoprecipitation, ChIP, flow cytometry, and more, and are also applicable for several other research areas including Neuroscience, Immunology, Stem Cell research, and Cancer research.

BioLegend's reagents are supported by 100% product guarantee, superior customer service, and a quality management system that is certified by ISO 13485:2003. Our aggressive product development program is supported through internal hybridoma development, technology licensing, collaborations with the scientific community, and in-house product validation and testing.

To view all our Cell Biology product categories, visit: biolegend.com/cell_biology

BioLegend Tools and Applications

Reagent Primary and Secondary Purified and Conjugated Antibodies

Low Endotoxin, Azide-Free Antibodies for Bioassays

Recombinant Proteins

Immunoassay Solutions

Chemical Probes and Cellular Assay Kits

MojoSort™ Magnetic Cell Separation

Applications For use in multiple applications including WB, IP, IHC, ICC, FC, ICFC, CyTOF®, ELISA, ChIP, or ELISPOT.

For use in functional assays such as cell activation, co-stimulation, blocking, or neutralization of cytokines.

For use in bioassays or as ELISA standards.

For quantification of single or multiple soluble analytes in standard ELISA using microplate reader or bead-based immunoassays suitable for flow cytometer.

Non-antibody based methods of detecting organelles or cell health based on chemical characteristics like hydrophobicity, charge, size and enzymatic activation.

For isolation and purification of cells from heterogeneous populations.

Web Link biolegend.com/cell_biology

biolegend.com/neuroscience

biolegend.com/immunobiology

biolegend.com/LEAF biolegend.com/recombinant_proteins

biolegend.com/bmia biolegend.com/cell_health_proliferation

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To ensure that we deliver the highest quality products, BioLegend’s cell bio antibodies undergo extensive validation processes which include:

• Testing on knockout (KO) or knockdown (KD) cell lysates (CRISPR/Cas9 or siRNA), and negative control cell lines to confirm antibody specificity

• Testing on multiple species (human, mouse, rat, yeast, bacteria, zebrafish) whenever applicable, to determine antibody cross-reactivity

• Side-by-side comparison testing with competitor’s antibody to ensure sensitivity

• Side-by-side comparison testing of lots with internal controls for lot-to-lot consistency

• Clones for one application (such as WB or ChIP) further validated in multiple applications (such as microscopy, flow cytometry) to provide the user the flexibility of supplementing their data with additional assays specific to their research

• Optimization of antibody suggested use by a titration range

Cell Biology Antibody Validation

Total lysates (15 µg protein) from 293T (lane 1) and 293T/HDAC2 knockdown (KD) cells (lane 2) were resolved by electrophoresis (4-20% Tris-Glycine gel), transferred to nitrocellulose, and probed with 1:500 diluted (1 µg/mL) Purified anti-HDAC2 Antibody, clone 13G8C67 (upper) or 1:3000 diluted Purified anti-GAPDH Antibody, clone poly6314 (lower). Proteins were visualized by chemiluminescence detection using a 1:3000 diluted goat anti-mouse-IgG secondary antibody conjugated to HRP for the anti-HDAC2 Antibody, and a donkey anti-rabbit IgG Antibody conjugated to HRP for anti-GAPDH Antibody. Lane M: Molecular weight ladder, M* indicates longer exposure.

Total cell lysate (15 µg protein) from MCF-7 (negative control), Jurkat and EL4 cells were resolved by electrophoresis (4-12% Bis-Tris gel), transferred to nitrocellulose, and probed with 1:500 diluted (1 µg/mL) purified anti-TCF12 antibody (clone W16168A) (upper). Proteins were visualized by chemiluminescence detection using 1:3000 diluted HRP goat anti-rat-IgG secondary antibody for clone W16168A (upper). Direct-Blot™ HRP anti-β-actin antibody (1:2000 diluted, clone 2F1-1) was used as a loading control (lower). Lane M: Molecular weight ladder.

Representative Data:

Negative Control Cell Line CRISPR/Cas9 Knockout

HDAC2 TCF12

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Total lysates (15 µg protein) from Raji (negative control), A549, and mouse brain, human brain and rat brain were resolved by electrophoresis, transferred to nitrocellulose, and probed with (A) 1:500 diluted (1 µg/mL) or (B) 1:250 diluted (2 µg/mL) purified anti-Dynamin-1 antibody (clone P83G4B6) (upper). Proteins were visualized by chemiluminescence detection using 1:3000 diluted HRP goat anti-mouse-IgG secondary antibody (Cat. No. 405306) for anti-Dynamin-1 antibody. 1:2000 (0.25 µg/mL) dilution of GAPDH (poly6314) antibody, or 1:1000 (0.5 µg/mL) dilution of β-actin (2F1-1) antibody was used as a loading control (lower). Lane M: MW ladder.

IHC staining of Formalin Fixed Paraffin Embedded (FFPE) normal human brain tissue. Following antigen retrieval using Sodium Citrate H.I.E.R (Cat. No. 928602), the tissue was incubated with anti-Dynamin-1 antibody (Clone P83G4B6) at 5 µg/mL overnight at 4°C. BioLegend’s Ultra-Streptavidin (USA) HRP kit (Multi-Species, DAB) (Cat. No. 929901) was used for detection followed by hematoxylin counterstaining, according to the protocol provided. Images were captured with a 40X objective.

IHC staining of Formalin Fixed Paraffin Embedded (FFPE) mouse brain tissue. Following antigen retrieval using Sodium Citrate H.I.E.R (Cat. No. 928602), the tissue was incubated with anti-Dynamin-1 antibody (Clone P83G4B6) at 5 µg/mL overnight at 4°C. BioLegend’s Ultra-Streptavidin (USA) HRP kit (Multi-Species, DAB) (Cat. No. 929901) was used for detection followed by hematoxylin counterstaining, according to the protocol provided. Images were captured with a 40X objective.

IHC staining of Formalin Fixed Paraffin Embedded (FFPE) rat brain tissue. Following antigen retrieval using Sodium Citrate H.I.E.R (Cat. No. 928602), the tissue was incubated with anti-Dynamin-1 antibody (Clone P83G4B6) at 5 µg/mL overnight at 4°C. BioLegend’s Ultra-Streptavidin (USA) HRP kit (Multi-Species, DAB) (Cat. No. 929901) was used for detection followed by hematoxylin counterstaining, according to the protocol provided. Images were captured with a 40X objective.

Validation of the same clone on multiple cell and tissue lysates, different applications, and different species for cross-reactivity.

Dynamin-1


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50 ng recombinant mouse OMG protein, mouse brain (M. brain) and rat brain (R. brain) total protein (15 µg each) were treated with or without 100 µg PNGase F at 37°C overnight to cleave the N-linked glycans from the proteins. All proteins were resolved by 4-12% Bis-tris gel electrophoresis, transferred to nitrocellulose, and probed with 1 µg/mL of purified anti-OMG antibody (clone A16003A). Proteins were visualized using a goat anti-rat-IgG secondary antibody conjugated to HRP and chemiluminescence detection. Direct-Blot™ HRP anti-β-actin antibody was used as a loading control.

4 replicate whole cell extracts (15 µg total protein) prepared from Jurkat cells treated with 2 mM H202 for 3 minutes were resolved by 4-20% Tris-Glycine gel electrophoresis and transferred to nitrocellulose membranes. Membranes were then treated with or without 30 units/mL of lambda protein phosphatase for 4 hours at room temperature. Each membrane was subsequently probed with 0.25 µg/ml (1:2000) purified anti-ZAP70 Phospho (Tyr493) antibody (Clone A16043C). Proteins were visualized by chemiluminescence detection using HRP goat anti-mouse-IgG. ZAP70 loading was confirmed using purified anti-ZAP70 antibody used at 0.25 µg/ml (1:2000 dilution).

Total cell lysates (15 µg protein) from Ramos cells treated without (lane 1) or with (lane 2) 2 mM H2O2 for 3 minutes, and Jurkat cells treated without (lane 3) or with (lane 4) the same stimulation were resolved by 4-20% Tris-Glycine gel electrophoresis, transferred to nitrocellulose, and probed with 1 µg/mL (1:500) purified anti-BTK Phospho (Tyr551)/ITK Phospho (Tyr512) antibody (Clone A16064A) or a competitor’s clone used at the manufacturer’s recommended concentration. Proteins were visualized by chemiluminescence detection using HRP goat anti-mouse-IgG (Cat. No. 405301) for Clone A16064A and HRP donkey anti-rabbit IgG (Cat. No. 406401) for the competitor’s clone. Membranes were then stripped and reprobed with an ITK antibody (Cat. No. 687302, 1 µg/mL, 1:500 dilution), and again stripped and reprobed with an antibody against BTK to confirm equal loading of the respective proteins.

Chromatin Immunoprecipitation (ChIP) was performed using commercial Protein-G coated 96 well high-throughput ChIP assay kit by loading 3 µg of cross-linked chromatin samples from HeLa cells starved overnight and treated with 10% FCS with either A) 1:50 dilution of Go-ChIP-Grade™ Purified anti-SSRP1 antibody (Clone 10D1), B) equal amount of Purified Mouse IgG2b, κ Isotype Control antibody, or C) competitor’s ChIP-grade Purified anti-SSRP1 antibody and D) equal amount of matched Isotype Control antibody as recommended by the manufacturer. The enriched DNA was purified and quantified by real-time qPCR using primers targeting human EGR-1 gene region. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the 5% of total amount of input chromatin.

Compared to competitor’s antibody side-by-side

Cell treatment optimization

BTK Phospho (Tyr551)/ITK Phospho (Tyr512)

OMG

SSRP1

ZAP70 Phospho (Y493)

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Total cell lysate (15 µg protein) from THP-1, HeLa and Raw264.7 cells were resolved by electrophoresis (4-20% Tris-Glycine gel), transferred to nitrocellulose, and probed with 1:250 (2 µg/mL), 1:1000 (0.5 µg/mL) and 1:2500 (0.2 µg/mL) diluted purified anti-SIRT5 antibody (clone O91G9) or competitor’s antibody used at manufacturer's recommended concentration (upper). Proteins were visualized by chemiluminescence detection using 1:3000 diluted HRP Goat anti-mouse IgG Antibody for clone O91G9 or 1:3000 diluted donkey anti-rabbit IgG Antibody conjugated to HRP for competitor’s antibody (upper). 1:2000 dilution of Direct-Blot™ HRP anti-β-actin antibody (clone 2F1-1) was used as a loading control (lower). Lane M: Molecular weight ladder, M* indicates longer exposure.

Jurkat cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes, and blocked with 10% FBS for 60 minutes. Cells were then intracellularly stained with an isotype control antibody at 2.0 μg/mL (1:250 dilution, Panel A), purified anti-Lck clone A17013D at 2.0 µg/mL (1:250 dilution, Panel B) and 1.0 μg/mL (1:500 dilution, Panel C), or purified anti-Lck clone LCK-01 at 2.0 µg/mL (1:250 dilution, Panel D) and 1.0 µg/mL (1:500 dilution, Panel E). Following incubation of all antibodies for 2 hours at room temperature, cells were incubated with Alexa Fluor® 594 goat anti-mouse IgG (Cat. No. 405308) at 2.0 µg/mL. Nuclei were counterstained with DAPI and the image was captured with a 60X objective.

Antibody suggested use optimization by titration

SIRT5

Lck


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Cellular organelles are membrane bound structures within a cell that have unique cellular functions. BioLegend offers a number of products to detect sub-cellular localization of proteins through organelle-specific dyes, or specific antibodies, that can be used for live or fixed-cell imaging, as well as for western blot and flow cytometry.

C57BL/6 mouse frozen cerebellum section was stained with anti-GFAP (clone 2E1.E9) Alexa Fluor® 647 (red), followed with Flash Phalloidin™ Green 488 (green).

Human paraffin-embedded cerebellum stained with Alexa Fluor® 647 anti-GFAP (red), Alexa Fluor® 488 anti-Tubulin Beta 3 (green), and DAPI (blue).

Cell Structure/ Organelles

Phalloidin is a very useful probe for imaging and stabilizing filamentous F-actin in fixed and permeabilized cells, providing structural and volumetric context to the cell.

GFAP is an intermediate filament protein that is expressed by numerous cell types of the central nervous system (CNS) including astrocytes and ependymal cells and is involved in the structure and function of the cell’s cytoskeleton.

Cytoskeleton:Specificity M P FP CP

Alpha-II Spectrin •BRST-2 •Centrin 2 (Caltractin) •Cytokeratin – 17 •Cytokeratin – 18 • •Cytokeratin – 19 •Cytokeratin – 7 •Cytokeratin – 8 •Cytokeratin-pan •Endoglin (CD105) • •Flash Phalloidin™ Green 488 •Flash Phalloidin™ NIR 647 •Flash Phalloidin™ Red 594 •GFAP • •Keratin – 10 • •Keratin – 6A •Keratin 1 •Keratin 14 •Keratin 14 •Keratin 15 •Keratin 5 •Kinesin Heavy Chain •MAP2 • •Myosin Heavy Chain •Myosin II – pan •Nestin • •Nestin Tail •Nestin, C-terminus •Nestin, Repeat Region •Neurofilament H & M (NF-H/NF-M) •Neurofilament H & M (NF-H/NF-M), Phosphorylated •

Neurofilament H (NF-H) • •Neurofilament H (NF-H), Phosphorylated • •Neurofilament L •Neurofilament M (NF-M) •Neurofilament Marker (pan neuronal cocktail) •NMHC II-C •Non-muscle Myosin Heavy Chain II-B •Vimentin • •α-Skeletal Muscle Actin •α-Smooth Muscle Actin •α-Tubulin •β-Actin • •β-Tubulin •β-Tubulin IIb (TUBB2) •β-Tubulin III (TUBB3) • • •γ-Tubulin •

Nuclear Markers:Specificity M P FP CP

7-AAD •Centrin 2 (Caltractin) •CytoPhase™ Violet •DAPI •DRAQ5™ •DRAQ7™ •Fibrillarin •Helix NP™ Blue •Helix NP™ Green •Helix NP™ NIR •Lamin A •mAKAP •Nuclear Pore Complex Proteins/NUP98 •Nucleolin-Phospho (Thr76/Thr84) •NUP153 •PCNA •Propidium Iodide •

Lysosome Markers:Specificity M P FP CP

CD63/LAMP-3 •CD107a (LAMP-1) •CD107b (Mac-3)/LAMP-2 •CD63/LAMP-3 •Cathepsin A • •Cathepsin B • •Cathepsin D • •Cathepsin E (CTSE) •LAMP 5 •

Mitochondrial Markers:Specificity M P FP CP

BAP37 •Cytochrome c •DJ-1 (PARK7) • •Grp75 (Mortalin) •HSD17B10 •HSP60 •LRRK2 •LRRK2 (NH2-terminus) •Mitofusin-1 •Mitofusin-2 •MitoSpy™ Green FM •MitoSpy™ Orange CMTMRos •MitoSpy™ Red CMXRos •Nhedc2 (NHA2) •Pancortin •PARIS (ZNF746) •PINK1 •Prohibitin •TFAM •UQCRC1 •VDAC1 •

M = Monoclonal Ab P = Polyclonal Ab FP = Functional Proteins CP = Chemical Probes

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HeLa cells were stained with anti-Lamin A Antibody (clone poly6135) followed by Alexa Fluor® 594 (red) conjugated goat anti-Rabbit IgG. Actin filaments were labeled with Alexa Fluor® 488 Phalloidin (green). Nuclei were counterstained with DAPI (blue).

NIH3T3 cells were stained with MitoSpy™ Red CMXRos (red), fixed and permeabilized with 1X True Nuclear™ Perm Buffer. Then the cells were stained with Flash Phalloidin™ NIR 647 (green) and counterstained with DAPI (blue).

HeLa cells were stained with 1 μg/ml Nuclear Pore Complex Proteins antibody (clone MAb414). Alexa Fluor® 594 (Red) Goat anti-Mouse IgG was used as secondary antibody. Nuclei were counterstained with DAPI (Blue).

Jurkat cells were treated with LEAF™ purified anti-human CD95, and stained with MitoSpy™ Red CMXRos following by staining with Alexa Fluor® 488 Annexin V.

MeOH fixed HeLa cells double stained with Clathrin (clone TD.1), and Giantin (Poly19243). Cells were counterstained with DAPI.

A431 cells were stained with anti-Keratin 5 antibody (clone Poly19055) and Alexa Fluor® 488 (Green) secondary antibody. Nuclei were counterstained with DAPI (Blue).

Lamin A is a member of the intermediate filament family, and is thought to function as a fibrous component of the nuclear lamina, providing a framework for the nuclear envelope, and possibly interacting with chromatin.

MitoSpy™ mitochondrial localization probes are cell-permeant, fluorogenic chemical reagents that are used for labeling mitochondria in living cells.

Nuclear pores are large protein complexes that cross the nuclear envelope and allow the transport of molecules across the nuclear envelope.

Clathrin is the most abundant protein in clathrin-coated vesicles that are involved in multiple vesicle trafficking pathways, and facilitate intracellular transport of cargo proteins following endocytosis.

Keratin 5 is a member of the type II (basic or neutral) cytokeratin family that are heteropolymeric structural proteins coexpressed during differentiation of simple and stratified epithelial tissues.

Ribosome:

Specificity M P FP CP

RPS6 •p90 Rsk •

Golgi:


Giantin •GPP130 •

Endoplasmic Reticulum:


GRP94 •PERK Phospho (Ser713) •

Centrosome Markers:


Aurora A (Aurora 2) •Aurora A (Aurora 2)-Phosphorylated (Thr288) •Aurora B •Ninein •Pericentrin •

Autophagosomes:


ATG5 •ATG17 •HSC70 •HSF1 •HSF2 •Hsp70 •Hsp90α •Hsp90α/β •Hsp90α/β •LAMP1 (CD107a) •LAMP2 (CD107B) •LAMP2 (CD107B) •LC3 •p62 (SQSTM1) •p62 • •Parkin •PINK1 •Ubiquitin •


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In developmental biology, cellular differentiation is the process where a cell changes from one cell type to another. These changes are largely due to highly controlled modifications in gene expression, and signaling cascades in the cell which include TGF-β/BMP signaling, Wnt/β-catenin signaling, Notch pathway, Hedgehog signaling, and Hippo signaling pathways. Cell differentiation not only occurs when a zygote transforms into a complex organism, but also continues into adulthood when adult stem cells fully differentiate into daughter cells during tissue repair and normal cell turnover. Because stem cells can replicate as well as differentiate to give rise to a variety of cell types, they are of considerable interest for potential medical applications.

Cell Development and Differentiation

Paraffin-embedded mouse testicle sections stained with anti-β Catenin 1 antibody (clone 12F7). Credit: Hans Snyder, Histologics.

Cell lysates from NTERA-2 cells (lane 1), NF-1 (lane 2), and HepG2 cells (lane 3) were probed with purified anti-SOX2 antibody (poly6519). Direct-Blot™ HRP anti-β-actin antibody (clone 2F1-1) was used as a loading control.

Recombinant Human SHH protein induces C3H10T1/2 differentiation in the presence of hBMP9 as measured by alkaline phosphatase production.

β-catenin plays a key role in Wnt signaling pathways and thus is involved in neural differentiation, synaptic plasticity, and neurodegenerative disease.

SOX2 is involved in the regulation of embryonic development and in the determination of cell fate.

Human Sonic Hedgehog (SHH) is expressed in embryonic tissues and is critical for the patterning of early embryos.

Specificity M P FP E LP

β Catenin 1 (CTNNB1) •Bax •B7-H1/PD-L1 • •Bcl-11b

BDNF •BMP-4,5,6,7,9,10,13,14 • •CD30 (TNFRSF8)-Fc Chimera • •CD30L • •CD117 (c-kit) • •CD133 •CD135 (Flt-3, Flk-2) • •CD349 (Frizzled-9) •CD90/Thy1 •c-MAF •DLL1 • •DLL4 •DHH •DKK-1 •Endoglin • •FGF-3,4, 19 • •FGF-6, 9,10, 17, 18, 21 •FOX3 (NeuN)

FOXA2 •FoxP3 •GATA3 •GFAP • •Gli-1 •Helios •HMGB1 • •IGFBP-1,6,7 • •IGFBP-3 • • •IGFBP-4 • • •IGFBP-5 •Ikaros •Jagged 1,2 •Lin-28A •Nanog •Nestin • •NKX2-1 (TTF-1) •Notch-1 •Notch-2 •Notch-3 •Notch-4 •

M = Monoclonal Ab P = Polyclonal Ab FP = Functional Proteins E = ELISALP = LEGENDplex™

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Human acute lymphoblastic leukemia cells HPB-ALL stained with PE anti- human Notch 4 (clone MHN4-2 PE).

Chromatin Immunoprecipitation (ChIP) was performed using NCCIT cells with either A) Go-ChIP-Grade™ Purified anti-Oct4 (Oct3) (clone 3A2A20), or B) equal amount isotype control antibody. The enriched DNA was purified and quantified by real-time qPCR using primers targeting human Sox2 gene regions.

Cell lysates from HeLa (negative control), HepG2 and NIH3T3 cells were probed with purified anti-Prox1 (clone W16098A) antibody, or loading control anti-GAPDH (poly6314) antibody. Lane M: molecular weight ladder.

Chromatin Immunoprecipitation (ChIP) was performed using Jurkat cells with either A) Go-ChIP-Grade™ purified anti-Bcl11b antibody, B) equal amount of isotype control antibody, or C) competitor’s ChIP-grade purified antibody, and D) equal amount of matched isotype control antibody as recommended by the manufacturer. The enriched DNA was purified and quantified by real-time qPCR using primers targeting human CDKN1A gene regions, which is known to be bound to Bcl11b.

Notch 4 is expressed by a variety of tissues including heart, lung, placenta, and liver, and controls many developmental processes. It functions as a receptor for transmembrane ligands such as the Jagged and Delta proteins.

Oct4 (Octamer binding transcription factor 4) is an important marker of the undifferentiated state and central regulator of pluripotency in ES cells. When embryonic stem cells are triggered to differentiate, Oct4 is downregulated, thus providing a model for the early events linked to somatic differentiation in the developing embryo.

Prox1 is a homeobox transcription factor responsible for progenitor cell differentiation and the development of several organs, such as those in the central nervous system and the liver.

Bcl-11b is a C2H2-type zinc finger protein that is expressed in thymus, mainly T cells. It is indispensable for T lineage development.Specificity M P FP E LP

Oct4 •p53 •p63 (TA) •p63 (ΔN) •Pax-2, 5, 6, 9 •PCNA •PLZF •Prox1 •PTEN •RUNX1 •Sca-1/Ly6A/E •SHIP-1 •SCF • • • • •Smad6 •Sonic Hedgehog •SOX2 • •SPI1 (PU.1) •SSEA-1, 3, 4, 5 •STAT1 •STAT1 Phospho (Ser727) •STAT3 • •STAT3 Phospho (Tyr705) •STAT5 •STAT5 Phospho (Tyr694) •STAT6 •STAT6 Phospho (Tyr641) •Syk •T-bet •TCF3 (E2A) •TGF-α • •TGF-β • • • •TMTSP (THSD1) •TNAP •TRA-1-60-R/Podocalyxin •TRA-1-81 •TRA-2-49 •TRA-2-54 •TSG •



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The progression of cells through cell division, cell growth, proliferation, and differentiation is regulated both by extracellular signals from the environment, as well as by internal signals that monitor and coordinate the various processes. An example of cellular regulation by extracellular cues is provided by growth factors that act through growth factor receptors on the cell surface. The effects of growth factors on the cell are carried down by several signaling pathways including the JAK/STAT, MAP-Kinase, and the PI3-Kinase pathways.

Growth Factors and Receptors

Standard curve generated using the LEGENDplex™ Human Growth Factor Panel.

Recombinant human IGFBP-4 binds human IGF-I in a dose-dependent manner (black circles). The binding of human IGF-I to human IGFBP-4 is blocked (purple circles) by increasing concentrations of Ultra-LEAF™ Purified anti-human IGFBP-4 antibody (Clone A15038E).

Recombinant human BMP-6 protein induces alkaline phosphatase production in the mouse chondrogenic ATDC5 cells.

The LEGENDplex™ Human Growth Factor Panel is a bead-based multiplex assay, using fluorescence-encoded beads suitable for use on various flow cytometers.

IGFBP-4 binds both insulin-like growth factors (IGFs) I and II, and prolongs the half-life of the IGFs, as well as alters their interaction with cell surface receptors.

Bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily and play a key role in embryonic development.


Activin A • •Amphiregulin • •Angiopoietin-2 • • •Artemin •Asprosin •BAFF • • •BAFFR • •BDNF •Betacellulin • •Betatrophin •BMP-4 •BMP-5 •BMP-6 •BMP-7 •BMP-9 •BMP-10 •BMP-13 •BMP-14 (GDF-5) •CD27L • •CD40L (TNFSF5) • • •CNTF •CRP • • •DHH •EGF • • • •EG-VEGF •Epigen •Epiregulin •EPO • • • •FGF-1-acidic • •FGF-3 • •FGF-4 • •FGF-6 •FGF-9 •FGF-10 •FGF-17 •FGF-18 •FGF-19 •FGF-21 •FGF-basic • • • •FLT3L •G-CSF • • •GDNF •GM-CSF • • • •HB-EGF •HGF • • • •


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ELISA assay using biotin anti-mouse LAP (TGF-β1) antibody (clone TW7-16B4) as the detection antibody to generate the example standard curve.

IFN-γ stimulated THP-1 (human monocytic cell line) stained with PE anti-human CD257 (BAFF, BLYS) Antibody (clone T7-241).

Staining of Clone NGFR5 on formalin-fixed paraffin-embedded normal human tonsil tissue.

Recombinant mouse M-CSF induces the proliferation of mouse myelogenous leukemia lymphoblast M-NFS-60 cells in a dose dependent manner (black triangles). Ultra-LEAF™ Purified anti-mouse M-CSF antibody (clone A16063L, purple circles) neutralizes the proliferation of M-NFS-60 cells induced by recombinant mouse M-CSF.

Recombinant human oncostatin M induced the proliferation of human erythroleukemic TF-1 cells in a dose dependent manner. BioLegend’s protein was compared side-by-side a competitor’s equivalent product.

TGF-β is a cytokine that has critical functions in the immune response by regulating Treg and Th17 cells.

BAFF, also known as B cell activating factor, tumor necrosis factor ligand superfamily 13B, B lymphocyte stimulator (BLYS), and TNF homolog, activates apoptosis, NF-κB, and JNK (THANK). BAFF is a type II transmembrane protein, and a member of the tumor necrosis factor ligand superfamily.

NGFR is expressed by many cell types including neurons and Schwann cells that promote cell apoptosis and regulate cell differentiation and neurogenesis.

M-CSF is a secreted glycoprotein that induces monocyte and macrophage colony formation from precursors in murine bone marrow cultures.

Human oncostatin M (OSM) was initially isolated from supernatant of U937 cells treated with PMA. It was identified by its property to inhibit the proliferation of A375 melanoma cells and other human tumor cells.


IGF-I • •IGF-II • •IGFBP-4 • • •Leptin • •LIF • • • •Light (TNFSF14) • •M-CSF • • •Midkine •Neurturin •NGF • •NGFR •NNT-1 (BCSF-3) •Noggin •NRG1 (Heregulin) EGF Domain (CF) •NT-3 •NT-4 •Oncostatin M • •OX40L • •PDGF-AA • •PDGF-BB • •PDGF-CC •PDGFRa, b • •Persephin •PLGF-1 •PTH • •Prolactin • •RANK (TNFRSF11A) • •RBP4 • • •S100A8/A9 Heterodimer • • • •SCF • • • • •Slit2-N •Sonic Hedgehog •TGF-α • •TGF-β • • • •Thrombopoietin (TPO) • •TNFSF18 (GITRL) • • •TRANCE (RANKL) • •TSLP • • • • •VEGF • • • •WISP-1 •WNT-7a •



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Cell growth and proliferation are among the most fundamental biological processes. The core cell cycle machinery that drives these processes have been conserved from yeast to humans. Proper spatial and temporal growth and proliferation is essential to allow normal development of organisms, and to maintain physiological homeostasis. Abnormal and uncontrolled cell proliferation leads to pathological conditions such as cancer.

Cell proliferation and viability can also be used to assess cell health. Different types of assays are used to detect these processes, such as using antibodies specific to proliferation and growth markers, measuring DNA synthesis, detecting metabolic activity, and/or cellular ATP/GTP levels.

Cell Proliferation, Growth, and Viability

Human FASL induced cytotoxicity in Jurkat cells is measured using both LDH-Cytox™ Assay Kit (OD 490nm) and Deep Blue Cell Viability™ Kit (RFU x 103).

Con A+IL-2-stimulated C57BL/6 mouse splenocytes stained with Ki-67 (clone 16A8) Brilliant Violet 421™ (filled histogram) or rat IgG2a, κ Brilliant Violet 421™ isotype control (open histogram).

Detection of Baf3/CCR3 cells viability using Resazurin fluorescence measurement. The increasing cell numbers correlate with the increasing fluorescence.

LDH-Cytox Assay™ Kit is a kit for determination of cytotoxicity by measuring lactate dehydrogenase activity released from damaged cells.

Nuclear protein Ki-67 plays an essential role in ribosomal RNA transcription and cell proliferation, and is strongly expressed in proliferating cells and has been reported as a prognostic marker in various tumors.

The Deep Blue Cell Viability™ Kit is formulated to study cell proliferation and quantification through the extent of resazurin reduction and resorufin production which is proportional to the number of metabolically active cells (live cells) present in the culture.

Specificity M P CP

ATPase Assay Kit •BrdU • •Calcein Violet-AM •Calcein-AM •CFSE Cell Division Tracker Kit •CytoPhase™ Violet •Deep Blue Cell Viability™ Kit •GTPase Assay Kit •Helix NP™ Blue •Helix NP™ Green •Helix NP™ NIR •Ki-67 •LDH-Cytox™ Assay Kit •p53 •PCNA •Tag-it Violet™ Proliferation and Cell Tracking Dye •TetraZ™ Cell Counting Kit •

M = Monoclonal Ab P = Polyclonal Ab CP = Chemical Probes

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Human peripheral blood mononuclear cells were stained with CFSE Cell Division Tracking Kit, and then stimulated with (filled histogram) or without (open histogram) PHA for 5 days. On day 5, cells were harvested and the CFSE fluorescent staining was analyzed by flow cytometry.

Mouse spleen 72 hours after adoptive transfer of Tag-it Violet™-labeled splenocytes (purple). Nucleated cells are stained using 25 µM DRAQ™ (red).

MOLT-4 cells fixed in 70% ethanol then stained with PE anti-human/mouse/rat PCNA Antibody (clone PC10).

Fresh (left) or day-old C57BL/6 splenocytes (right) were stained with Calcein Violet-AM and a cell-impermeant nucleic acid dye, SYTOX™ Red (colored). Black figure represents unstained splenocytes.

HeLa cells were fixed with 1% paraformaldehyde (PFA) for 10 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes, and blocked with 5% FBS for 30 minutes. Then the cells were intracellularly stained with 1 µg/ml anti-human/mouse/rat PCNA (clone PC10) Alexa Fluor® 594 (red) in blocking buffer, overnight at 4°C, followed by Alexa Fluor® 488 Phalloidin (green) staining for 20 minutes. Nuclei were counterstained with DAPI (blue).

Tag-it Violet™ Proliferation and Cell Tracking Dye can be used for cell tracking and for proliferation assays.

CFSE (formally known as 5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester of CFDA SE) is widely used for cell proliferation assays and in vivo cell tracking.

Calcein Violet-AM is a fluorogenic, cell-permeant fluorescent probe that indicates cellular health by detecting the activity of nonspecific esterases.

Proliferating cell nuclear antigen also known as PCNA or the DNA polymerase δ auxiliary protein, is a 36 kD trimeric ring that acts as a DNA-polymerase sliding clamp expressed in the nucleus of all proliferating cells.


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The cell cycle is a series of precisely timed and carefully regulated stages in a cell that consists of cell growth, DNA replication, distribution of the chromosomes to daughter cells, and cell division. It consists of four distinct phases: Go (resting) /G1 (post-mitotic gap) phase, S (synthesis) phase, G2 (growth) phase, and M (mitosis) phase. A large number of proteins are involved in the complex machinery that regulate and maintain the cell cycle such as cyclins, cyclin-dependent kinases, tumor suppressors, and checkpoint proteins. These proteins ensure that the cell cycle continues only after the completion of a prior stage. In the event of a DNA damage, checkpoint proteins are activated and checkpoint-arrested cells resume cell-cycle progression once the DNA damage has been repaired. Cells with irreparable DNA lesions undergo permanent cell-cycle arrest or apoptosis.

Cell Cycle

C57BL/6 mouse thymus cells were fixed using chilled ethanol and stained with Helix NP™ NIR.

HeLa cells were intracellularly stained with anti-Aurora B antibody (clone W16153A) followed by Alexa Fluor® 594 (red) conjugated goat anti-rat IgG (Cat. No. 405422). Nuclei were counterstained with DAPI (blue). The images displayed HeLa cells at different phases of mitosis.

Helix NP™ NIR is a far-red emitting nucleic acid stain that can be used to stain fixed cells for cell cycle analysis.

Aurora B plays a critical role in cell division and chromosome-microtubule interactions during mitosis. The expression level of Aurora B is dramatically increased during the S/G2/M phases of the cell cycle.

Specificity M P CP

Alpha B Crystallin •APC7 •Artemis •Aurora A (Aurora 2) •Aurora A (Aurora 2)-Phosphorylated (Thr288) •Aurora B •BrdU • •C2H2 zinc finger protein phospho linker region (HpTGEKP)

•C/EBPα •Centrin 2 (Caltractin) •Cdc2 (p34) •Cdk2 •Cdk4 •CDK5

CDK7 Phospho (Ser164/Thr170) •CDKN1A •CDKN2A •Centrin 2 •CFSE Cell Division Tracker Kit •Chk2 •ch-TOH •c-Myc •Cyclin A •Cyclin B1 •Cyclin D1 •Cyclin D2

Cyclin D3 •CytoPhase™ Violet •DAPI •DNA-PKcs Phospho (Thr2609) •DRAQ5™ •DRAQ7™ •Eg5 •


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Total lysates from 293T (Lane 1), 20 nM scrambled siRNA (lane 2), 5 nM (Lane 3)and 20 nM (Lane 4) Centrin 2 siRNA treated 293T cells were probed with purified anti-Centrin 2 antibody (clone W16110A). Direct-Blot™ HRP anti-β-actin Antibody (clone 2F1-1) was used as a loading control.

Ramos cells loaded with BrdU for 1.5 hours (left) or left as no load controls (right) stained using the Phase-Flow™ FITC BrdU Kit and DAPI.

HeLa cells were stained with purified anti-CDKN1A (clone W15115A) antibody, followed by Alexa Fluor® 594 secondary antibody (red), Alexa Fluor® 488 conjugated Phalloidin (green), and DAPI (blue).

Ramos cells treated with 5 µM CytoPhase™ Violet dye for 90 minutes at 37°C. Cells were then acquired on a flow cytometer equipped with a 405 nm laser with a 450/50 bandpass filter.

WB analysis of UV untreated and treated HeLa cells probed with Purified or Direct-Blot™ HRP anti-H2A.X Phospho (Ser139) antibody (clone 2F3). Purified anti-GAPDH antibody (clone Poly6314) was used as a loading control.

Centrins are small calcium binding proteins and the Centrin-2/Rad23B/XPC complex acts as an essential component of the nucleotide excision repair (NER) pathway.

BrdU is a Uridine derivative and a structural analog of thymidine, which can be incorporated into DNA during the S-phase of the cell cycle as a substitute for thymidine.

Cyclin-dependent kinase inhibitor 1A (CDKN1A), also known as P21 and Cip1, is a potent cyclin-dependent kinase inhibitor. CDKN1A binds to and inhibits the activity of cyclin-cyclin-dependent kinase2 or kinase4 complexes, thus functions as a regulator of cell cycle progression at G1.

CytoPhase™ Violet is a cell-permeant DNA-binding dye that can be used for flow cytometric analysis of cell cycle in live or fixed cells, and also for counterstaining the nuclei of cells in fluorescent microscopic imaging.

H2A.X becomes phosphorylated on serine 139 after double-stranded DNA breaks. Phosphorylated H2A.X (γ-H2AX) promotes DNA repair and maintains genomic stability.

Specificity M P CP

Eg5-Phosphorylated (Thr927) •Flightless-I Protein •H2A.X • •H2A.X Phospho (Ser139) •Helix NP™ Blue •Helix NP™ Green •Helix NP™ NIR •KIN-28 •MAD2 •MCM3 •MCM5 •MCM6 •MCM7 •Ninein •NOD1 •p27Kip1 •p53 •p97/VCP •PARP •Phospho-CAK (cdk7) (Ser164/Thr170) •PLK-1 •PLK-1 Phospho (Thr210) • •PRC1 •Prohibitin •Propidium Iodide •PTEN •Rad23B •RCC1 •SMC1L1/SMC1 •Stathmin/Op18 Phospho (Ser16) •TPX2 •TRF2 •



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Cell signaling is the transmission and integration of signals from the outside to the inside of the cell. These signals are chemical, molecular, or mechanical cues that exert a specific effect on the cell. Cell proteins called receptors bind to signaling molecules (ligands) to initiate a physiological response, and different receptors are specific for different ligands. Once a receptor binds its ligand, it undergoes a conformational change, which in turn initiates a series of biochemical reactions within the cell. These intracellular signaling pathways (also known as signal transduction cascades) amplify the message using a network of enzymes and proteins. Enzymes in these signaling cascades typically modify the proteins they interact with in a process known as post-translational modification (PTM). Phosphorylation is one of the most common PTMs for regulating the activity and function of proteins. Since the ability of cells to correctly process signals is critical for their survival, errors in cell signaling typically cause diseases such as cancer, diabetes, and autoimmunity.

Cell Signaling

Jurkat cells treated without (Lane 1) or with (Lane 2) 2 mM H202 for 3 minutes were probed with purified anti-ZAP70 Phospho (Tyr493) antibody (Clone A16043C). Equal ZAP70 loading was confirmed using purified anti-ZAP70 antibody.

HeLa cells were were intracellularly stained with anti-CARD9 Antibody (clone 13D9C60) followed by Alexa Fluor® 594 (red) conjugated goat anti-mouse IgG. Actin filaments were labeled with Alexa Fluor® 488 Phalloidin (green). Nuclei were counterstained with DAPI (blue).

ZAP70 is a member of the Syk protein tyrosine kinase subfamily, expressed exclusively in T cells and NK cells that plays an essential role in T cell receptor (TCR) signaling in combination with the Src family kinases, LCK and FYN.

CARD9 is an adaptor protein that mediates the signaling downstream of C-type lectin receptors (CLRs), such as Dectin-1, Dectin-2, and Mincle.

Specificity M P

β Catenin 1 (CTNNB1) •β-Arrestin-1/2 •14-3-3 ζ/δ •14-3-3 ε •ABCA3 •Aggrecan •AKT1 •AKT2 •Akt Phospho (S473) •Allergin-1 •Amphiregulin •Arginase I •ATF4 •Aurora A (Aurora 2, AIK) •Aurora A (Aurora 2, AIK) phosph, (Thr288) •Aurora B •B-Raf •BTK Phospho (Tyr223) •CAK (Cdk7) (Ser164/Thr170) •CaMKII •CARD9 •CASK •cdc2 (p34) •CDK5 •c-Met •c-REL •DDX17 (p82) •DDX17 (p82, p72) •DDX58 •DNA-Pkcs Phosphorylated (Thr2609) •Dopamine D3 receptor •DR3 (TNFRSF25) •DUX4 •E1 Ubiquitin Activating Enzyme •EGF •EGFR Phosphorylated (Tyr1068) •eIF2α •ER81 •ERK1 •ERK1/2 •ERK1/2 Phospho (Thr202/Tyr204) •ERK2 •FOXP1 •FOXP3 Delta 2 (exon 2 deleted) •Fyn •GAB1 •

Specificity M P

GATA3 •GSK-3α •Hamartin (TSC1) •IκB-α •IKBKB (IKKβ) •IKKα •IKKγ (NEMO) •Lck •LCK Phospho (Tyr505) •LKB1 (STK11) •Lyn •MAPKAP1 •MERTK •mTOR •MyD88 •NLRP 1, 2, 7, 12 •NOD1 •NOS2 •Notch1β •NTAL •NTAL (LAT2) •P2RX7 •p38 MAPK Phospho (Thr180/Tyr182) •p90 Rsk •PERK Phospho (Ser713) •Phosphotyrosine •PIK3R1 •PIR-A/B •PKCα Phospho (T638) •PLCγ-1 •PLK-1 •PLK-1 - Phosph, (Thr210) • •Polyubiquitin (K63-linkage) •PTEN •PYK2 •RAPTOR •RPS6 •SH2D1A (SAP) •SHIP-1 •SIK2 •SIRT1 •SMAD6 •SNAP-25 •SOCS3 •Sonic Hedgehog (SHH)

SOX17 •SPHK1

M = Monoclonal Ab P = Polyclonal Ab

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MCF7 cells were stained with purified anti-PTEN (4C11A11) antibody, followed by staining with DyLight™ 488 conjugated goat anti-mouse IgG (green) antibody. Nuclei were stained with DAPI (blue).

HeLa (Lane 1), 5 nM (Lane 2), and 20 nM (Lane 3) TSC2 siRNA treated HeLa cells were probed with purified anti-TSC2 antibody. Direct-Blot™ HRP anti-β-actin Antibody was used as a loading control.

Human peripheral blood lymphocytes were stimulated with (filled histogram) or without (open histogram) recombinant human IL-4, permeabilized with True-Phos™ Perm Buffer, and intracellularly stained with STAT6 Phospho (Tyr 641) (clone A15137E) PE/Cy7.

HeLa cells were intracellularly stained with SMAD6 Antibody (P85H3) and followed by Alexa Fluor® 594 goat anti-mouse IgG (red). Actin filaments were labeled with Alexa Fluor® 488 Phalloidin (green). Nuclei were counterstained with DAPI (blue).

NIH3T3 cells (lane 1), PC3 cells (lane 2), and HeLa cells (lane 3) were probed with purified anti-PTEN antibody (clone 4C11A11) antibody. Purified anti-β-actin antibody (poly6221) was used as a loading control.

PTEN is a tumor suppressor with lipid and protein phosphatase activity that negatively regulates the PI3K/AKT pathway.

Tuberous sclerosis 2 (TSC2) forms the tuberous sclerosis complex with TSC1 and functions as a crucial negative regulator of the Rheb/mTOR pathway.

The Tyr641 residue of STAT6 is phosphorylated by Jak. Phosphorylated STAT6 forms homodimers, transclocates to the nucleus, and regulates transcription of target genes.

SMAD proteins are TGF-β signaling components that consist of receptor-regulated SMADs (SMAD1/2/3/5/9), a common SMAD (SMAD4), and inhibitory SMADs (SMAD6/7). SMAD6 is a negative regulator of TGF-β and bone morphogenetic proteins.

Specificity M P

STAT 1 , 3, 4, 5, 6 •STAT 2 •STAT1 Phospho (Ser727) •STAT3 Phospho (Ser727) •STAT3 Phospho (Tyr705) •STAT6 Phospho (Tyr641) •STING •SWAP70 •Syk •Syntrophin •TAB1 •TICAM-2 (TRAM) •TIGIT (VSTM3) •TIMP-1 •TRAF 1 , 3, 6 •TRIM •TSC2 •Ubiquitin •Wntless •XCR1 •ZAP70 •ZAP70 Phospho (Tyr292) •ZAP70 Phospho (Tyr319) •ZAP70 Phospho (Tyr319)/Syk Phospho (Tyr352) •ZAP70 Phospho (Tyr493) •

C57BL/6 mouse frozen brain section was stained with anti-CaMKII (clone 6G9) antibody and anti-GFAP (clone Poly28400) antibody followed by Alexa Fluor® 594 (red) conjugated goat anti-mouse IgG and Alexa Fluor® 488 (green) conjugated goat anti-mouse IgG. The nuclei were counterstained with DAPI (blue).

The Ca2+/calmodulin (CaM)-dependent protein kinases (CaMKs) are multifunctional serine/threonine kinases whose activities are regulated through Ca2+ signaling.


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In eukaryotic cells, DNA is assembled into chromatin with repeating units of nucleosomes. Each nucleosome consists of eight core histone proteins, two each of H2A, H2B, H3, H4, which is wrapped with 147 base pairs of DNA. This complex forms the basis of the epigenetics landscape and is a dynamic system that undergoes multiple post-translational modifications (PTMs) and interacts with several regulatory proteins, including transcription factors to control gene expression. Common examples of PTMs are methylation, acetylation, phosphorylation, and ubiquitination that can alter chromatin structure. This, in turn, can dictate gene expression patterns in a cell by regulating the relative accessibility of transcription factors and the transcriptional machinery to the DNA.

Epigenetics and Transcriptional Regulators

Chromatin Immunoprecipitation (ChIP) was performed with chromatin samples from HeLa cells with either A) Go-ChIP-Grade™ Purified anti-Histone H4 Trimethyl (Lys20) Antibody (clone 6F8-D9), B) equal amount of matched isotype Control Antibody (clone MOPC-21), or C) competitor's ChIP-grade Purified anti-Histone H4 Trimethyl (Lys20) Antibody and D) equal amount of matched Isotype Control Antibody as recommended by the manufacturer. The enriched DNA was purified and quantified by real-time qPCR using primers targeting human MYOD gene regions.

HeLa cells were stained with anti-IRF1 Antibody (clone 13H3A44) followed by Alexa Fluor® 594 (red) conjugated goat anti-mouse IgG. Actin filaments were labeled with Alexa Fluor® 488 Phalloidin (green). Nuclei were counterstained with DAPI (blue).

NTERA-2 cells were intracellularly stained with anti-Nanog (clone 16H3A48) Alexa Fluor® 647 (filled histogram) or mouse IgG1, κ Alexa Fluor® 647 isotype control (open histogram).

Lysine N-methyltransferase 5C (KMT5C) is a Histone-lysine N-methyltransferase that specifically trimethylates Histone H4 Lysine 20 (H4K20). The more heavily methylated H4K20 becomes, the less transcription of the associated genes occurs.

IRF1 was the first identified member of the interferon regulatory transcription factor family, and is involved in the regulation of cell cycle progression, tumor suppression, and apoptosis.

Nanog is a homeodomain-containing transcription factor that is essential for early embryonic development.

Specificity M P

Ahr •Aiolos •AND-1 (WDHD1) •Ataxin-3 •ATF4 •ATF7 •BACH 1, 2 •BATF •Bcl11b •Bcl-6 •Blimp-1 •BRD4 •C/EBP b •C/EBP b (2 isoforms C/EBP b, LAP) •C/EBPa •C2H2 zinc finger proteins •c-Fos •c-MAF •c-REL •CRP •Cystatin C •DNMT1 •DNMT3B •DUX4 •EGR2 •eIF-2a •eIF3D •eIF3F •eIF3G •EIF3I •eIF3J •Elongin B •Elongin C •EOMES •EOS •ER81 •ETS2 •FOXA2 •FOXD3 •

Specificity M P

FOXO1 •FOXO3 •FOXP1 •FOXP3 •FOXP3 Delta 2 •GAB1 •GATA3 •GCN5 •Gli-1 •Granzyme B •Gre A •Gre B •HDAC1 • •HDAC 2, 3, 4, 7, 8 •HIF1-beta •Histone H2B •Histone H3 •Histone H3 (C-terminus) •Histone H3 Dimethyl (Lys9) •Histone Lysine Demethylase (NO66) •Histone H3 Monomethyl (Lys9) •Histone H3 Nonmethyl (Lys9) •Histone H4 Monomethyl (Lys20) •Histone H3.1 Phospho (Ser28) •Histone H3 Trimethyl (Lys9) Antibody •Histone H4 Trimethyl (Lys 20) •HIV TAT •HOXA3 •Hoxb-1 •HRF •Ikaros •IRF1, 2, 3, 4, 5,6, 7, 8, 9 •KIN-28 •LEF1 •MAFB •MBD1 •Nanog •NFATc1, c2, c3 •

M = Monoclonal Ab P = Polyclonal Ab

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Staining of the seminiferous tubules in formalin fixed paraffin embedded human testis tissue using DUX4 (clone P2B1/DUX4).

Chromatin Immunoprecipitation (ChIP) was performed using chromatin samples from HeLa cells starved overnight and then treated with IL-6 with either A) Go-ChIP-Grade™ Purified anti-STAT3 Phospho (Ser727) Antibody (Clone A16089B), B) equal amount of Isotype Control Antibody, or C) competitor’s ChIP-grade Purified anti-STAT3 Phospho (Ser727) Antibody and D) equal amount of matched Isotype Control Antibody as recommended by the manufacturer. The enriched DNA was purified and quantified by real-time qPCR using primers targeting human IRF1 gene region.

Staining of anti-Histone H3 Trimethyl (Lys9) Antibody (Clone 6F12-H4) on frozen rat brain.

Non-treated and Tunicamycin (Tm) treated HeLa and 293T were probed with purified anti-ATF4 (clone W16016A) antibody or competitor's antibody. Direct-Blot™ HRP anti-β-actin (clone 2F1-1) antibody was used as a loading control.

Double homeobox 4, also known as DUX4, is a protein that has been reported to function as a transcriptional activator of paired-like homeodomain transcription factor 1 (PITX1).

Stat3 transcription factor is activated by phosphorylation at Tyr705, which induces dimerization, nuclear translocation, and DNA binding. It is involved in the activation of genes required for cell growth and apoptosis.

Histone H3 can be modified by phosphorylation, acetylation, ubiquitination, ribosylation, and methylation, that regulate gene expression.

ATF4 belongs to the family of basic zipper-containing proteins that regulate two important target genes: CHOP (transcription factor C/EBP homologous protein) and GADD34 (growth arrest and DNA damage–inducible 34).

Specificity M P

NFIL3 •NF-kappaB p100/p52 •NF-kB p50 •NF-kB p65 • •NF-κB p105/p50 •NF-κB p65 •NKX2-1 •NR4A2 •NR5A2 •Nur77 •NusA •OCT 2, 4 •Pax-2 •Pax-5 •Pax-6 •PAX9 •POU2AF1 •PPAR-γ •PLZF •Prox1 •RAP30 •RARα •RBPJ •RNA Polymerase beta •RNA Polymerase beta Prime •RNA Polymerase II •RNA Polymerase II RPB1, 3, 4, 5, 6, 8,11 •RNA Polymerase TFIIB •RNK •RORg •RORgt •RORα •RUNX1, 2, 3 •SATB1 •Sigma 32 •Sigma 54 (strain MG1655) •Sigma 70 (strain MG1655) •Sigma E •

Specificity M P

Sigma F (strain MG1655) •Sigma FecI •Sigma S (Strain MG1655) •Sir1 •Sir3 •SOX17 •SOX2 • •SOX2 (NH2 terminus) •Sp3 •SP4 •SPI1 •Spt16 (FACT140 complex) •SSRP1 •STAT1 Phospho (Ser727) •STAT 1 , 3, 4, 5, 6 •STAT1 Phospho (Ser727) •STAT3 Phospho (Ser727) •STAT3 Phospho (Tyr705) •STAT6 Phospho (Tyr641) •TAL1 •Tata Binding Protein •T-bet •TCF1 •TCF1 (TCF7) •TCF12 •TCF3 •TCF8 •TDRD3 •TFIIB •Th-POK •TICAM-1 •TIF1b •TOX •TTF-1 •XBP-1s •ZBP-1 •


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Cells adhere to each other and to the extracellular matrix through complex cellular structures that involve many proteins from receptor molecules to structural scaffolding proteins. These connections include focal adhesions (cell to matrix), adherens junction (cell to cell), and tight junctions (impermeable cell to cell connection). Transmembrane glycoproteins called cell adhesion molecules (CAMs) are integral to the formation of adhesions. Some CAMs are Ca2+ dependent such as cadherins, selectins, and integrins, whereas others such as N-CAM, which is expressed by a variety of cell types including most nerve cells, are Ca2+ independent. Cellular adhesion not only links cells, but can be involved in transmitting downstream signal as well.

Cell Adhesion and Extracellular Matrix

WB analysis of HUVEC and HUVEC treated with recombinant Human TNF-α probed with different concentrations of BioLegend’s (clone A16047A) or competitor’s anti-CD106 (VCAM-1) antibody, and Direct-Blot™ HRP anti-β-actin Antibody as a loading control.

Human colon carcinoma cell line (HT29) stained with purified CD324 (clone 67A4) (filled histogram), or purified mouse IgG1, κ (open histogram) followed by anti-mouse IgG FITC.

Staining of Integrin α2 (clone P1E6) on frozen normal human kidney tissue.

CD106/VCAM-1 is involved in cell adhesion and acts as a counter-receptor for VLA-4 (α4/β1 integrin) and LPAM-1 (α4/β7 integrin).

E-Cadherin/CD324 is a member of the cadherin superfamily and is a calcium-dependent, transmembrane cell-cell adhesion glycoprotein.

CD49b/ Integrin α2 associates with CD29 (β1 integrin) to form VLA-2, a collagen and laminin receptor on many cell types including activated T cells, neuronal cells and epithelial cells.

Specificity M FP E LP

ADAM22 •α-E-Catenin •Aggrecan • •AMIGO-1 •β Catenin 1 (CTNNB1) •Brevican •Cadherin 11 •Cathepsin A • •Cathepsin B • •Cathepsin D • •Cathepsin E •CD11a •CD11a/CD18 (LFA-1) •CD11b •CD11c (Integrin αx subunit) •CD18 (Integrin β2) •CD29 (Integrin β1) •CD31 •CD34 •CD41 •CD43 •CD43 Activation-Associated Glycoform

•CD47 •CD48 •CD49a •CD49b (Integrin α2) •CD49C (Integrin α3, URO-1) •CD49D (Integrin α4) •CD49e (Integrin α5) •CD49f •CD50 (ICAM-3) •CD51 (Integrin αvβ5) •


CD51/61 •CD54 (ICAM-1) • •CD58 •CD61 •CD62E (E-selectin) • •CD62P (P-selectin) •CD62L •CD62P (P-selectin) •CD66a •CD66a/c/e •CD66C (CEACAM 6) •CD73 •CD102 (ICAM-2) •CD103 (αE Integrin) •CD104 •CD105 •CD106 (VCAM-1) • •CD107b (LAMP-2) •CD138 •CD144 (VE-Cadherin) •CD146 •CD147 (EMMPRIN) •CD151 (PETA-3) •CD155 (PVR) •CD156c (ADAM10) •CD161f •CD169 (Sialoadhesion) •CD170 (Siglec-5) • •CD171 •CD172a (SIRPα) •CD207 (Langerin) •CD209a (DC-SIGN) •CD227 (Muc-1) •

M = Monoclonal Ab FP = Functional Proteins E = ELISALP = LEGENDplex™

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Immobilized recombinant mouse E-Selectin/CD62E protein induces 85-100% adhesion of U937 cells. BioLegend’s protein was compared side-by-side to a competitor’s equivalent product.

IHC staining of purified anti-Connexin 43, 360-382 antibody (clone P2C4) on formalin-fixed, paraffin-embedded normal human brain tissue.

IHC staining of anti-Brevican antibody (clone N294A/6) on formalin-fixed paraffin-embedded mouse brain tissue. Ultra Streptavidin (USA) HRP Detection Kit (Multi-Species, DAB, Cat. No. 929901) was used for detection followed by hematoxylin counterstaining.

E-Selectin, an adhesion molecule, belongs to the selectin/LECAM family and is only expressed in endothelial cells.

Connexins are a family of transmembrane proteins that assemble to form vertebrate gap junctions, thus also called Gap Junction Proteins.

Brevican is a brain chondroitin sulfate proteoglycan, specifically expressed in the central nervous system, that plays a role in development and formation of the brain extracellular matrix and may be involved in malignancy of brain tumor cells.


CD324 •CD324 (E-Cadherin) •CD325 (N-Cadherin) •CD326 (Ep-CAM) •CD370 (CLEC9A, DNGR1) •CD371 (CLEC12A) •Claudin-1 •CLEC-2 • •Clusterin • •Connexin 43 •EphA2 • •Ephrin-A1 • •ESAM •FAK •γ Protocadherin A (pan reactive) •γ-protocadherin-B2 •Galectin-1 •Galectin-3 • •Galectin-4 •Galectin-9 • •HVEM • •ILK •Integrin α4β7 •Integrin α9β1 •Integrin αV/β3 •Integrin β4 •Integrin β5 •Integrin β7 •LFA-1 •LPAM-1 (Integrin a4b7) •Mac-2 (Galectin-3) •MAdCAM-1 •MMP-1 • •


MMP-2 • • • •MMP-3 • • •MMP-7 •MMP-8 • •MMP-9 • • • •MMP-10 •MMP-12 •Neuroligin-1 •Neuroligin-3 •NrCAM •Oligodendrocyte-myelin glycoprotein •p120 Catenin •Paxallin • •PNAd •Podoplanin • •Siglec E •Siglec H • •Siglec-3 •Siglec-9 •SALM2 (LRFN1) •SynCAM4 •TIMP-1 • • •TIMP-2 • • •TIMP-3 •URO-1 •Urokinase (uPA) • •VAP-1 • •Vimentin •Vitronectin • •


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Apoptosis (also referred to as programmed cell death) is a naturally occurring process that leads to changes in cell morphology and ultimately cell death. It is a highly regulated and sequential process that can be initiated through one of two pathways - the intrinsic apoptosis pathway, where the cell kills itself because of factors such as stress and DNA damage, and the extrinsic pathway, where the cell kills itself because of external signals from other cells. Although both pathways induce cell death by activating caspases, the intrinsic pathway is mainly regulated by the Bcl-2 family of proteins, whereas the extrinsic pathway is under the control of members of the TNFR family of proteins which are the death receptors.

Unlike apoptosis, necrosis is an unregulated process of cell death that is caused by factors external to the cell or tissue, such as trauma, toxins, or infection. Cell death by necrosis is characterized by swelling of the cell, nuclear shrinkage, breakage of the nucleus into fragments, and plasma membrane rupture.

As cells die, whether by apoptosis or necrosis, impermeant nucleic acid stains, esterase activity-dependent probes, and mitochondrial respiration probes become useful tools to detect the live/dead status of the cells.

Cell Death

Human T-cell leukemia cell line, Jurkat, treated (left) or non-treated (right) with LEAF™ purified anti-human CD95 (clone EOS9.1) mAb, then stained with FITC Annexin V Apoptosis Detection Kit with 7-AAD.

Apoptotic cell death induced by recombinant human FASL protein in Jurkat cells.

Annexin V (or Annexin A5) is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells.

Fas ligand (FasL, CD95L, TNFSF6) is a type-II transmembrane protein that belongs to the tumor necrosis factor (TNF) family. Its binding with its receptor induces apoptosis.

Specificity M P FP E LP CP

7-AAD Viability Staining Solution •AIM2 •Akt Phospho (Ser473) •AKT1 •AKT2 •Alix •Annexin V •Arginase I • •ASC (TMS-1) •ATG17 •BAD •BATF •Bax • •BCL10 •Bcl-2 •Bcl-XS/L •BID •Bin1 •Bromodeoxyuridine (BrdU) • •Calcein-AM •Calcein Violet-AM •CARD9 •Caspase 11

Caspase-1, 2L, 7, 8, 9, 10, 12 •Caspase-3 •Cathepsin B, D • •CFSE Cell Division Tracker Kit •c-Myc •CXCL12 (SDF-1β)

Cytochrome c •Daxx •DcR1 (TRAIL-R3, CD263) •DcR3 •DDX3X •DDX5 •DR3 (TNFRSF25, TRAMP) •DR4 (TRAIL-R1, CD261) •DR5 (TRAIL-R2, CD262) •

M = Monoclonal Ab P = Polyclonal Ab FP = Functional Proteins E = ELISALP = LEGENDplex™CP = Chemical Probes

biolegend.com

25

Human acute lymphoblastic leukemia cell line MOLT-4 were stained with Bax (clone 2D2) Alexa Fluor® 488 (filled histogram) or mouse IgG1, κ Alexa Fluor® 488 isotype control (open histogram).

HeLa cells were treated with 20% EtOH and stained with Zombie Violet™ (magenta, left), Zombie Green™ (green, middle), or Zombie Red™ (red, right) and then fixed with 1% PFA. Nuclei were counterstained with DRAQ5™ (blue).

HeLa cells were stained with anti-Cytochrome c (clone 6H2.B4) Alexa Fluor® 594 overnight and Phalloidin Alexa Fluor® 488 (green). Nuclei were counterstained with DAPI (blue).

The relative levels of pro-apoptotic proteins such as Bax and anti-apoptotic proteins such as Bcl-2 determines whether cell death will occur following an apoptotic stimulus.

Zombie dyes are a family of amine-reactive fluorescent dyes that is non-permeant to live cells but permeant to cells with compromised membranes. Thus, it can be used to assess live vs. dead status of mammalian cells in microscopy and flow cytometry.

Cytochrome c initiates apoptosis by binding Apaf-1, which activates procaspase 9.


7-AAD Viability Staining Solution •AIM2 •Akt Phospho (Ser473) •AKT1 •AKT2 •Alix •Annexin V •Arginase I • •ASC (TMS-1) •ATG17 •BAD •BATF •Bax • •BCL10 •Bcl-2 •Bcl-XS/L •BID •Bin1 •Bromodeoxyuridine (BrdU) • •Calcein-AM •Calcein Violet-AM •CARD9 •Caspase 11

Caspase-1, 2L, 7, 8, 9, 10, 12 •Caspase-3 •Cathepsin B, D • •CFSE Cell Division Tracker Kit •c-Myc •CXCL12 (SDF-1β)

Cytochrome c •Daxx •DcR1 (TRAIL-R3, CD263) •DcR3 •DDX3X •DDX5 •DR3 (TNFRSF25, TRAMP) •DR4 (TRAIL-R1, CD261) •DR5 (TRAIL-R2, CD262) •


Fas (CD95) • • •Fas-L (CD178) • • •Galectin-1 •Galectin-3,9 • •Galectin-4 • •Granzyme A • • •Granzyme B • • • •Granzyme K •H2A.X • •H2A.X Phospho (Ser139) •Helix NP™ Blue •Helix NP™ Green •Helix NP™ NIR •LDH-Cytox™ Assay Kit •MitoSpy™ Green FM •MitoSpy™ Orange CMTMRos •MitoSpy™ Red CMXRos •NLRP1 •NOD1 , 2 •P62 • •PARP •Perforin • •Propidium Iodide Solution •sTNF-RI (TNFRSF1A) •sTNF-RII (TNFRSF1B) •Survivin •TL1A (TNFSF15) •TNFα • • • •TNFβ • • •TRAIL (TNFSF10) • •TWEAK (CD255) • •TWEAK receptor (Fn14, CD266) •Zombie Dyes •

HeLa cells, untreated (-) or treated with Staurosporine (+, 1 µM, 8 hours) probed with anti-PARP (clone 5A5) antibody. Direct-Blot™ HRP anti-β-actin was used as a loading control.

PARP (Poly (ADP-ribose) polymerase) is a nuclear protein that functions in base excision repair, poly(ADP-ribosyl)ation of acceptor proteins involved in chromatin architecture and DNA metabolism, and participates in protein modification to enhance or repress transcription.


26

Normal Cell

Normal Cell

Tumor Cell

Dormant Tumor Cell

Necrotic Tumor

ImpairNK, CD8+ T Cell,γ/δ T Cell Response

Impair NK, CD8 T Cell Response

MICA/B

HLA-G,E

Soluble CD83

Angiogenesis

VEGF, M-CSF, IL-1β,IL-6

Soluble PDL1 Reduce T Cell Proliferation

Impair T Cell E�ectors

Immunosuppression

Fas Ligand

Apoptosis of Tumor-Speci�c E�ectors

Epigenetic SilencingLoss of IFN-γR Signaling

CarcinogensInherited DefectsRadiationVirusesChronic In�ammation

Cancer TherapiesAdaptive T Cell TransferMonoclonal AntibodiesAntibody Drug ConjugatesDCs/Peptide Vaccines

Immune Exhaustion or InhibitionTumor-Cell VariantsGenetic and Epigenetic ChangesResistance to Immune DetectionTumor Maintained Chronically

Rae-1(Mouse)MICA/B(Human)

Genetic Instability and /or Immune Selection

Danger SignalsUric AcidECM ProductsHeat Shock ProteinsType 1 IFNs

Immune EditorsIFN-α/βIFN-γIL-12TNF-αPerforinTRAILChemokines

CD4+T Cell

CD4+T Cell

CD8+T Cell

CD8+T Cell

CD8+T Cell

Dendritic Cell

Dendritic Cell

NKCell

NKT Cell

M1 Macrophage

γ/δT CellTCR

??

NKG2Dγ/δT Cell

Tumor Microenvironment

Bone Marrow

Release of MDSC into Peripheral Blood

(Tumor Derived)

MDSC Maturation

Di�erentiation

Recruitment

Recruitment

Lymph Nodes

MDSC

MDSC

VISTA

MDSC

MDSC

MDSC

M2 Macrophage

pDC(Plasmacytoid DC)

iDC(Immature DC)

Immature DC

TGF-βIL-10

pSTAT3FOXP3

MDSCCMPHPC

NOS2ROSRNOSArg1

MDSC Dendritic Cell

T Cell Deletionor Anergy,Apoptosis

T Cell Deletionor Anergy, Apoptosis

IL-4IL-13GM-CSF

IL-4IL-13GM-CSFTGF-β

IL-4IL-13

Arg1 IL-4IL-10IL-13TGF-β

B7-1

B7-1

B7-1

CTLA-4

T E�ectorApoptosis

IL-6VEGFM-CSFIL-1βPGE2

IL-6VEGF

GM-CSFCSF-1

CCL2CCL12CXCL5

IL-2IL-10IFN-α

NOS2NORNOS

Arg1NOS2NORNOS

COX2PGE2

Arg1R-NOSL-Arginine

Tumor Cell

Tumor-DerivedFactors

Treg CD8+T Cell

CD8+T Cell

Galectin

Tumor-DerivedSoluble FactorsIDO

MHC Class I MoleculeIFN-γ ResponseCCL27(Cutaneous Tumors)CD80PDL1

NKCell

Tregs in Tumor Escape CheckpointsTregs in Tumor Escape Checkpoints

Tumor Sculpting

Immunosuppression

Immunosuppression

Tumor Escape

E�ector Cell(NK Cell, Monocyte, Macrophage)

TRAIL

CD91

IL-12

CD4+T Cell

CD8+E�ector T Cell

CD8+T Cell

Proliferating Cancer Cells

Help

FasL

Fas

FcR

CD4+ T CellDi�erentiation Dendritic Cell

CD8α+Dendritic Cell

PDL1

PDL1

PDL1

PD-1

(Molecular Shield)

E�ector Cell Elimination, Anergy, Apoptosis

Angiogenesis

Partial Tumor Destruction

PolyploidTumor Cell

Other “Eat Me” Signals

ADCC

KillingKilling

Killing

Phagocytosis

TRAILR

TRAILR

TRAILR

TRAIL

Fas

FasL

IL-12

IL-12

IL-12R

TCR

CD1d

TRAIL

NKT Cell

NK Cell

NOSROS

IL-1TNF-α

IL-2

IL-4IL-5IL-6IL-10IL-13

CD36L

Calreticulin

LDLReceptorrelated protein

CD36

TCR

MHCII

TCR

CD28CD137

GITR

OX40

MHCI

ανβ5L

ανβ5

M1 Macrophage

Th1

Th2

IFN-γ

IFN-γ

IFN-γ

IFN-γ

IFN-γTNF-α (Cytostatic)

IFN-γ

IFN-γ

IFN-γ

IFN-γ IFN-γR

Tumor-Reactive Antibodies

(β-actin, HSP, Cytokeratin)

ImmunosuppressionImmunosuppression

Tumor Destruction(Host Protection)Tumor Destruction(Host Protection)

Tumor Antigen

Opsonization, Complement, ADCC

Oxidative BurstRelease of IL-1, TNF-α, Cytotoxicity

Tumor AntigenPresentation

AntigenPresentation

Dendritic Cell

Dendritic Cell

Granulocyte

Macrophage, Dendritic Cell Engul�ng HSP-Peptide ComplexVia CD91

Perforin Granzyme

Tumor AntigenCross-Presentation

NKG2D

NKG2DLs

Tumor-In�ltrating B Cell

04-0017-01

CD8+Treg

Th3/Tr1

Treg

APC

APC

CD80/CD86

CD73

CD39ATP

AMP

Adenosine

AdenosineReceptor

AdenosineReceptor

Enhanced TregActivity

CD80CD86

CTLA4

+

IDOCCL22

IDOSOCS1Arginase

IDO

IDO

Tryptophan

Immunosuppression

TGF-βIL-10

PGE2

GangliosidesGalectin1/9

E�ector T Cell

E�ector T Cell

Antigen Uptake

FOXP3

Treg

Treg

IL-10TGF-βIDOCOX2PGE2Immunosuppressive Molecule

APC Dysfunction/T-Cell Anergy

IL-12MHCCD80CD86

CD4+CD25+CTLA4+GITR+

IL-10TGF-β

T CellApoptosis and Anergy

MetabolizedTryptophan

pDC

T E�ector

Macrophage, Dendritic Cell(Tolerogenic)

+

+

+

pSTAT3VEGFLXR-L

IL-13TGF-β

CD4+CD25T Cell

Cancer Recognition/EliminationCancer Recognition/Elimination Shedding of Tumor Recognition Antigens Shedding of Tumor Recognition Antigens

MDSC-Induced ImmunosuppressionMDSC-Induced Immunosuppression

Treg

HSP-Peptide Complex

The 3 E’s of Cancer ImmunoeditingThe 3 E’s of Cancer Immunoediting

Contact BioLegendUS & Canada Toll-Free: 1.877.246.5343 (877-BIOLEGEND)International: 1.858.768.5800Fax: 1.877.455.9587email: [email protected], [email protected]

US Headquarters:San Diego, CA 92121

CD205CD209CD273MHC Class II

CD11bCD11cCD80CD83CD86

CD49bCD56CD122CD314 (NKG2D)CD335 (NKp46)

CD4CD183Tim-3T-bet

CD4CD193CD194CD294Tim-1

CD4CD25FOXP3CD127low

Activation:GARPLAP (TGF-β)

Human:CD33CD11bCD14neg

Mouse:CD11bGR-1

Dendritic Cell Natural Killer Cell Th1 Cell Th2 Cell Treg Cell MDSC

biolegend.comWe would like to thank Dr. Robert Schreiber of Washington University - School of Medicine for his contributions to this poster.

Interactive Poster: biolegend.com/cancerimmunoediting

©BioLegend, Inc. 2014

Cancer Immunoediting

Death Receptor 6(TNFRSF21)

TWEAK-R (TNFRSF12A/Fn14)

Decoy Receptor 2 (TNFRSF10D/TRAIL-R4)

Zombie DyesPropidium Iodide (PI)7-Amino-Actinomycin (7-AAD)

Decoy TRAIL Receptor R1 (TNFRSF23)Decoy TRAIL Receptor R2 (TNFRSF22)Decoy Receptor 1 (TNFRSF10C/TRAIL-R3)

Decoy Receptor 3 (TNFRSF6B)

TWEAK (TNFSF12)

TWEAK (TNFSF12)

TRAIL (TNFSF10)

PI

BAT1, TIM-4

CD36

TLR RIPK1

RIPK3

Necrosome

RIPK1

PKR

MLKL

FLIP

DAI

Necrosulfonamide

IFN

JAK-STAT

Virus

TRIFCaspase 8

NLRC4

Cytosolic Bacteria

Cholera toxin B,Gram-negative bacteria

Intracellular LPS

FlagellinType 3 SecretionSystem Rods/Needles

Caspase 4, 5, 11

Caspase Oligomerization

Pyroptosis

Pore formationCell swellingLysis

HMGB1ATPIL-1αLactose dehydrogenase

?

NLRP3

ASCASC FocusFormation

AIM2

Procaspase 1

In�ammasome

Caspase 1

IL-1β, IL-18

FADD

FADDTRADD

Necrostatin 1

De novotranscription

Bacterial DNA

Death Receptor

Extracellular ATP Bacterial pore-forming toxins Monosodium urate crystalsCholesterol crystals

oxLDL

ICAM3?CD14

MER

GAS6

7-AAD

Sphingosine-1-Phosphate

NucleotidesATP, UTP

T

C

G

A

P2y2

PANX1

Sphingosine-1Phosphate Receptor

Lysophosphocholine

G2A?

Phosphatidylserine

TRAIL (TNFSF10)

TRAIL (TNFSF10)

RAIDD

Caspase-2 PIDDosome

PIDD-CC

APAF1

AIF, Endo G

smac/diabloHtra 2/Omi

Target Molecule Cleavage(Actin, DNA, Nuclear Lamins)

Bax, BakCytochrome C

BH3 Only Protein Activation

Bax, Bak Oligomerization

Bcl-2 Bcl-2-xL, MCL-1

p53

SevereDNA Damage

TRAIL (TNFSF10)

FAS-L(TNFSF6/CD178)

FAS-L (TNFSF6/CD178)

Osteoprotegerin (TNFRSF11B)

Phosphatidylserine

Phosphatidylserine (PS)

Phospholipids

Caspase 3,7

CDC50

Actomysin Contraction

ROCK1

Annexin V

NF-kB

Zombie DyesNH2

NH2

Ca2+

Lactoferrin

CD31

CD46CD47

Neutrophil Recruitment

FAS (TNFRSF6/CD95)

Death Domain

Death Inducing Signaling Complex (DISC)

TNF RI (TNFRSF1A)TNF RII (TNFRSF1B)

Death Receptor 4 (TNFRSF10A/TRAIL-R1)Death Receptor 5 (TNFRSF10B/TRAIL-R2)

Death Receptor 3 (TNFRSF25, APO-3)

TNF-α

TRAF1

TRAF2

FADD

FADD

FADD

FADD

Procaspase 8,10

Procaspase 8,10

Caspase 8,10

Caspase 3

CAD

ICAD

ICADDegradation

Caspase 9

Caspase 3

Caspase 6

Xkr8Scramblase

ATP11CFlippase

Caspase 7

XIAP

XIAP

XIAP

Procaspase 3

Procaspase 6

Procaspase 7

Procaspase 2

Caspase 2

Procaspase 9

Membrane PermeabilityDisruption

ROS GenerationDNA Fragmentation

Procaspase 3

Bid

t-Bid

TRADD

TRADDTRADD

TRADD

RIP1

RIP1

CIAP1/2K63 Ubiquitination

K63 UbiquitinRemoval

RIP1

FLIP

RIP1

TRAF5

TRAF 3

?

Bcl-2 Superfamily Caspases

Keep-out SignalKeep-out Signal

Don’t Eat Me SignalsDon’t Eat Me Signals

Eat-Me SignalsEat-Me Signals

Outer Lea�et PS ExposureEat-Me Signal

Outer Lea�et PS ExposureEat-Me Signal

Find-Me Signals Find-Me Signals

Endoplasmic Reticulum StressDNA DamageSurvival Factor Loss

Chemotaxis, EngulfmentChemotaxis, Engulfment

Membrane Rupture Membrane Rupture

IntracellularIntracellular

ExtracellularExtracellular

BlebbingBlebbing

Cytoskeletal Rearrangement, EngulfmentCytoskeletal Rearrangement, Engulfment

DNA FragmentationDNA Fragmentation MitochondriaMitochondria

ApoptosomeApoptosome

Apoptotic CellApoptotic Cell

PhagocytePhagocyte

Cell SurvivalProliferationCell SurvivalProliferation

04-0051-00


Mechanisms of Cell Death

biolegend.com

Macrophage Monocyte NeutrophilDendritic Cell Fibroblast Mast Cell

Pro-apoptotic Anti-apoptoticBax Bid BCL-2 A1Bok BikBak

BCL-XL BLFL-1

Bim

Noxa BCL-W BCL-B

Bad

Puma MCL-1

InitiatorCaspase 2 Caspase 3 Caspase 1Caspase 8 Caspase 6 Caspase 4Caspase 9 Caspase 7 Caspase 5Caspase 10

P


US Headquarters:San Diego, CA 92121Interactive Poster: biolegend.com/celldeath

We would like to thank Dr. Douglas Green, St. Jude Children's Research Hospital, for his contributions to this poster.

Bacteria

Goblet Cells Paneth Cells

Injury

Cell Proliferation,Injury Repair

MucusReplenishment

MucusReplenishment

Macrophage

Macrophage

Macrophage

Di�erentiation

Activation TNF-α, NO

TNF-α / NO (Nitric Oxide)-Producing Cell

MonocyteRecruitment

Dendritic CellRecruitment

NK Cell Recruitment

CCL2, 3, 5, 7

IL-1β, IL-12, IL-18, TNF-α

NK Cell

IL-12

IFN-γ

Virus

Fungi

Parasite

ParasiteBacteria

COX2

PGE2

IL-13

IL-25 IL-33TSLP

MucusProduction

Innate Lymphoid Cell

Eccrine Gland

Eccrine Epithelial Cells

Maturation

TNF-α,Histamine

IgE-MediatedDegranulation

AntigenPresentation/Costimulation

Distribution ThroughSweat: Cathelicidins,Dermcidin

Injury

Injury

Bacterial Lysis

Desquamation

ROS, ATP

IL-1β, TNF-α

Mast Cell

CD11cCD80 CD86 MHC II CLRs NLRs TLRs

CD23 CD117 CD203c FcεRIα

CD11b CD15 CD66c Mouse:Gr-1

CD3- CD11b- CD11c- B220- CD49b-

CD45 CD90 CD127

CD49b CD56 CD122 CD314 (NKG2D) CD335 (NKp46) CD336 (NKp44)

AAM:Arginase Ym-1 RELMαMouse:F4/80

Classic:CD68 CD14 CLRs NLRs TLRs

CD3- CD11b- CD19- CD49b-

Human:CD123 CD303Mouse:B220 Ly6C CD317

β-Defensins

Allergens

Allergens

Pathogen Killing and Shedding, Barrier Function

In�ammation,Anti-MicrobialResponses

Pathogen Elimination

ROS/NOS PhagosomalDigestion

Phagocytosis

Phagosome Phagosome

Apoptosis

TLRs, NLRs, CLRs, Other Intracellular Receptors

TLRs, NLRs, CLRs, Other Intracellular Receptors

Antigen Presentation,Costimulation, IL-12

Dendritic CellMaturation

Dendritic Cell

ROSNOS

IL-13

MucusProduction

Glycans

Lipids

Dendritic Cell

Dendritic Cell

Dendritic Cell

IL-13

Larvae Control

Innate Lymphoid Cell

MucusProduction

Eosinophils,Basophils, Mast Cells

Antigen Presentation

Alarmins,IL-4, TSLP

ROS/NOS

CLRs and TLRs

TLRs and FcεRs

CXCL8 (IL-8)/KCCCL3

CLRs , TLRs

LarvaeControl

ROSNOS

Chitins, Proteases, Secreted Products

IL-25 IL-33TSLP

?

?

Alternative Activated Macrophage (AAM)Arginase+, Ym-1+, RELMα+

Neutrophil Recruitment

Neutrophil

Degranulation

Pathogen Killing

α-DefensinsCathepsinsElastaseLysozyme

IL-6

CX3CL1

PerforinGranzyme B

IFN-γ

Dendritic Cell

Dendritic Cell Plasmacytoid Dendritic Cell (pDC)

Natural Killer (NK) Cell Macrophage Mast Cell Neutrophil Innate Lymphoid Cell

Mature Dendritic Cell

Antigen Presentation and Activation of Adaptive Immunity

Other “Eat Me” Signals

Tumor Control

NK CellActivation

NK Cells

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

TumorGrowth Inhibition

pDC Maturation/Activation

Regulatory FunctionsModulation of Cytokine ProductionDendritic Cell Maturation

NK Cell Activation Anti-Viral Functions

Infected Cell Killing



IFN-α

Virus

IFN-α

TLR7, 9

Fibroblast

RLR, DNA Sensors

Tumor Killing

Tumor Cells

NET (Neutrophil Extracellular Trap)

Pathogen Containmentand Damage

MucinsAnti-Microbial Moleculesα-DefensinsAngiogenin-4Reg3γ

Innate immune mechanisms of cancer promotionInnate immune mechanisms of cancer promotion

Th2 and Th22ResponseTh2 and Th22Response

Th2 ResponseTh2 Response

Cytotoxic T LymphocytesCytotoxic T Lymphocytes

Th1 and Th17 ResponseTh1 and Th17 Response

04-0029-00

Tumor -AssociatedMacrophages (TAM)

Tumor Cell Survival and Proliferation

Myeloid-DerivedSuppressor Cell (MDSC)

Inhibition ofTumor Proliferation

CCL3,4,5,20CXCL12

TNF-α IFN-γActivation

IL-12IL-15IL-18

αvβ5LCD36LTRAIL-R

αvβ5CD36TRAIL

NKp44, 46TRAILNKG2DNKG2DNKp30

? TRAIL-R MULT1 MICA/B B7-H6


Innate Immunity

biolegend.comTLR: Toll-like receptorNLR: NOD-like receptorCLR: C-type lectin receptorRLR: RIG-I-like receptor


US Headquarters:San Diego, CA 92121Interactive Poster: biolegend.com/innateimmunity

We would like to thank Dr. Ruslan M. Medzhitov of the Yale School of Medicine for his contributions to this poster.

Blood-Brain Barrier

Neural Stem Cells

Neural Stem Cells

Complement Proteins

InjurySite Injury

SiteInduce In�ammation &Immune Cell RecruitmentIL-8, MCP-1, IL-6, IL-1β, RANTES

MCP-1, RANTES, IL-6, IL-17A, IL-17F, IFN-α

Protein aggregates Acute Brain InjuryGenetic Risk Factors Aging Pathogens

IL-10, TGF-β,IL-4, IL-13

Axonal Remodeling Neural RepairNeurotrophic Factor Release

Debris Clearance Via Phagocytosis

Misfolded/AggregatedProteins, Cell Debris

Migration towards lesionNeurogenesis Neuronal Replacement

MMP-9, TNF-α, MCP-1, IL-1β, IL-8

Pro-apoptotic CytokinesIFN-γ, TNF-α

IFN-γ, TNF-α

IFN-γ,TNF-α

MMP-2, MMP-9, ROS

IL-8, MCP-1, MIP1α & β, RANTES

IL-6, TNF-α, IL-1β

PericyteEndothelial Cells

AngiogenesisNeovascularizationECM ReconstructionSDF1, VEGF,MCP-1, IGF1, FGF2

SDF1, VEGF,MCP-1, IGF1, FGF2

Reactive Astrocyte

Reactive Astrocyte

Astrocyte

Reactive Astrocyte

Reactive AstrocyteReactive

Astrocyte

Reactive Astrocyte

Astrocyte/Glial Scar

Arg1: ECM Reconstruction & Tissue Repair

AstrocyteNeuron

Neuron Neuron

Complement Sheddingand Activation

Diseased Neuron

Protein Aggregate

Complement Binding

Complement Binding

AutoantibodyBinding

Microglia & AstrocyteActivation

ComplementExpression & Secretion

Neuron

Autoantibodies

Circulating Autoantibody

Circulating Autoantibody

Cytotoxic Cytokines

Complement

Chemotaxis,Phagocytosis, Cell Lysis

Microglia

Pro-in�ammatory Anti-in�ammatory

Rami�ed State

T Cell T Cell

T Cell

Cytokines,Chemokines (CCL2)

T Cell

Invading Pathogens

B Cell

B Cell

B Cell B Cell MonocyteMonocyte

Monocyte

Monocyte

Monocyte

Synapse

CRCR

CR

C1q

C3b

C3

Protein X

MCP-1, IL-6, TNF-α, IL-1β, IL-17A, IL-17F

Oligodendrocytes

Neurotrophic Support

Anti-in�ammatoryCytokines

Cytokines

Reactive Microglia

Reactive Microglia

Reactive Microglia

Reactive Microglia

Reactive Microglia

Reactive Microglia

Reactive Microglia

Reactive Microglia

Reactive Microglia

Reactive Microglia

Reactive Microglia

Oxidative StressOxidative Stress

ApoptosisApoptosis

Suppression of NeurogenesisSuppression of Neurogenesis

BBB Disruption Immune Cell In�ltrationBBB Disruption Immune Cell In�ltration

Activated T CellActivated T Cell

Neuron DeathNeuron Death

Neuron DeathNeuron Death

04-0079-00


Neuroin�ammation

biolegend.com

DAMPs(Damage-associated Molecular Patterns) DAMPs(Damage-associated Molecular Patterns) PAMPs(Pathogen-associated Molecular Patterns) PAMPs(Pathogen-associated Molecular Patterns)

T Cell ResponsesT Cell Responses

T Cell ResponsesT Cell Responses

Astrocyte ProliferationAstrocyte Proliferation

Neuron SurvivalNeuron Survival

Pro-in�ammatoryPro-in�ammatory Anti-in�ammatoryAnti-in�ammatory


US Headquarters:San Diego, CA 92121Interactive Poster: biolegend.com/neuroin�ammation

We would like to thank Prof. Dr. Michael Schlossmacher of the University of Ottawa, Canada for his contributions to this poster.

Complimentary Posters & BrochuresView, Download, and Request: biolegend.com/literatureCancer Immunoediting

Neurodegeneration

Innate Immunity

Mechanisms of Cell Death

Neuroinflammation

Dendritic Cells, Monocytes and Macrophage Biology

biolegend.com

27

04-0088-00


Autophagy

biolegend.com

Autophagy in Axonal TransportAutophagy in Axonal Transport

Retrograde Axonal Transport

Autophagosome

Kinesin

Cargo

Cargo

LysosomeFusion with the lysosome in the soma

Dynactin

Dyneinhtt HAP1

Autophagosome

KinesinDynactin

Dynein polyQ-httHAP1

-Impairment of Retrograde Transport-Defects in Cargo Delivery

Damaged NeuronDamaged Neuron

Healthy NeuronHealthy Neuron


US Headquarters:San Diego, CA 92121Interactive Poster: biolegend.com/autophagy

We would like to thank Dr. Charbel Moussa of Georgetown University Medical Center, Washington, D.C. for his contributions to this poster.

Chaperone-mediated Autophagy (ATG Independent)Chaperone-mediated Autophagy (ATG Independent)

Parkinson’s DiseaseParkinson’s Disease

Healthy CellHealthy Cell

LysosomeLysosome

LAMP-2A

Mutant α-Synuclein

α-Synuclein

Mutant LRRK2

α-Synuclein Aggregate

Oligomers at Lysosome

α-Synuclein Oligomers at Lysosome

Inhibition of CMA SubstrateDegradation

Inhibition of LAMP-2A Multimerization & CMA Substrate Degradation

Destabilization of Lysosome Membrane

Tau Aggregation in the Cytosol

Mutant Tau

Degradation ofLRRK2 and α-Synuclein

hsc70

hsc70

Co-chaperones

Co-chaperones

LRRK2

Autophagy Events in the BrainAutophagy Events in the Brain

Site of:• Autophagosome Formation• Predominant Localization of Lysosomes• CMA-mediated Protein Degradation

SomaSoma

Site of:• Autophagosome Formation and Maturation during Retrograde Axonal Transport• Clearance of Synaptic Vesicles, Proteins, and Receptors

AxonAxon

Involved in Macroautophagy-mediated:• Clearance of Protein Aggregates• Synaptic Pruning

AstrocytesAstrocytes

Site of: • Macroautophagy-mediated Neurotransmitter Receptor Degradation

DendritesDendrites

Involved in: • Autophagy-induced Cell Death• Synaptic Pruning

MicrogliaMicroglia

Site of Macroautophagy-mediated:• Clearance of Protein Aggregates• Increased Myelination

OligodendrocytesOligodendrocytes

Mitophagy (Selective Macroautophagy)Mitophagy (Selective Macroautophagy)

Damaged MitochondriaLoss of Membrane Potential

PINK1 Accumulation

Phagophore

Phagophore

LC3LC3

LC3LC3NDP52

TAX1BP1

NBR1 p62

Poly-UbChains

TBK1 TBK1

Optineurin

LC3Rab7

Fis1

TBC1D15TBC1D17

LC3

LC3

LC3

Autophagosome Formationand Fusion withthe Lysosome,Acidi�cation& Degradation

Parkin Recruitment, Phosphorylation &Activation

Ubiquitin Phosphorylation

Ubiquitination of Mitochondrial Proteins by Parkin (e.g. VDAC, Mitofusin 1/2)

OMMTOM TOM TOMTIM TIM

PARL PARLMPP MPP

IMM OMM

IMMSubstrate

Damaged MitochondriaDamaged Mitochondria

Damaged MitochondriaDamaged Mitochondria

Healthy MitochondriaHealthy Mitochondria

Activation and Recruitment of MacroautophagyMachinery

P P

P

P

P

P

P P P

P

PINK1PINK1

Dimerization &Autophosphorylation

ProteasomeDegradation

Cytosol

MutantParkin

MutantPINK1

Macroautophagy Process (ATG Dependent)Macroautophagy Process (ATG Dependent)Stress Signal Stress Signal

ULK1/2 Kinase Complex Activation

AgingProtein Aggregateetc.

ULK1/2 P

FIP200 ATG13

Initiation Initiation

VPS34 Complex Activation Beclin1

P

VPS34 ATG14L

PI3P Generation

VPS15

Endoplasmic Reticulum Endoplasmic Reticulum

Nucleation Nucleation

Precursor Vesicles

Phagophore Elongation

ATG9

ATG9

ATG9

Golgi Network Golgi Network

Expansion Expansion

Phagophore

PI3P

PI3P

PI3P

PI3P

DFCP1

WIPI1/2

WIPI1/2

DFCP1 PI3P

PI3P, WIPIs, and DFCP1 Recruitment

LC3-II PE

ATG12 ATG5

ATG16LE3

Binding to Cytosolic Cargo

ATG5 Complex Recruitment

Autophagosome

Autophagosome

Lysosome Lysosome

Autolysosome

Maturation Maturation Fusion with the Lysosome

Membrane Fusion

Lipidation with LC3-PE

LC3 LC3-I

Cleavage

ATG7 E1

ATG3 E2

ATG4

Aggrephagy (Selective Macroautophagy)Aggrephagy (Selective Macroautophagy)

Misfolded Protein

Lysosome

Autolysosome

Protein Aggregate

Ubiquitin

UbiquitinatedProtein Aggregate

Autophagy Receptors(e.g. p62, ALFY, Optineurin, NBR1)

ATG5 Complex

Phagophore Formation

LC3-II

PE

Autophagosome

* Mouse only

TRAF6TRAF6

TRAF6

EndocytosedTLR4

TLR7/8

TLR13

TLR9

ssRNA

Unmethylated CpG DNA

Unmethylated CpG DNA

dsRNA

TLR3

TRIFTRIF

TRAM RIP1

RIP1

23srRNA

TIRAP

Pro�lin

TLR12Homodimer

TLR11/12Heterodimer TRAF3

TRAF3

TRAF3

IKKεTBK-1

ProteasomalDegradation

Ubiquitin

Ubiquitin

RICK/RIP2

RICK/RIP2

Procaspase-1

Procaspase-1

Caspase-1

Pro IL-1βPro IL-18

IL-1β IL-18 In�ammationPyroptosis

NLRP3

TXNIP

TXNIP

Active State

TRX

TRX TXNIPTRX

ROS

Cathepsins

Crystalline Structures

Pannexin-1

P2X7

PAMPs NOX Enzymes

NOD1

NOD2

Ubiquitin

IKKβ

IKKβ

NF-κB

NF-κB

NF-κB

NF-κBActivation

MAPKActivation

NF-κB

NF-κB

NF-κB

IKKα

IKKβ

IKKγ/NEMO

IKKα

IKKα

AP-1 Activation

TBK1

TBK1

pol IIIViral DNA

Viral RNA

STING

IntracellularBacteria

Proin�ammatoryFactors

Cyclic Dinucleotides

NEMO

IRAK1 IRAK2

IRAK4

LPS

TIRAPMyD88

MyD88

MyD88

?

Lipopeptides

Flagella

TLR5

TLR10TLR2

TLR2

TLR1/6

CD14

CD14

TLR4/MD2

TRAF6

IRAK1 IRAK2

IRAK4IRAK1 IRAK2

IRAK4

TAB2/3

TAB2/3MuramylDipeptide

Endocytosed BacteriaDAMPsPore-forming toxinsCalcium In�ux?

γ-D-glutamyl-mesodiaminopimelic acid

TAK1

TAK1

TAB1

TAB1

TAB2/3

IRF7

ASC

AIM2

ASC

Foreign dsDNA

JAK1

JAK1

Tyk2

Tyk2

STAT1

STAT2

TRF9

ISGF3

IFNStimulatedGenes

Type 1 IFNs

IFNAR1IFNAR2

MAVS Aggregation

Dimerization

TRIM25Riplet RNF135

K63 Polyubiquitination

Heterotetramer Formation

RIG-I

IRF7

Type 1IFNs

IRF3

IRF3

IRF3

IL-15RANTESCXCL10

IL-1βIL-6IL-10IL-16iNOSTNF-αGM-CSFMIP-1αMIP-1βMCP-1RANTES

TAK1

TAB1

c-JunJNK

p50p60

RelA

c-Fas AP-1

AP-1

AP-1

CREB

RAS

Dectin-1

DC-SIGN

Dectin-2

Mincle

β-glycan

SYK

FcγR

FcγR

α-Mannose,Cord Factor

High Mannose,α-Mannans

High Mannose,Fucose

SYK

SYK

RAF-1

NLRP3 Activation

CARD9

ROS

MALT1Bcl-10

ptd Ins (3, 4, 5) P3

ptd Ins (4, 5) P2

PKC

BTK

RAC

DAGRHO

p38

PKC

InsP3

PLCγ

PLCγ

PI3K

SYKLyn

Antigen

ITAM

IgG Antibodies

FcγRs

α subunit

γ2subunit

SOS

RAS

CGAS

Mda5

IPS-1

ERKJNK

ADCCPhagocytosisCytokine ReleaseOxidative Burst

Ca2+

Ca2+Export

CalcineurinNFAT

NFAT

CREB

p38/MAPK

ParasitesParasites

BacteriaBacteria

BacteriaBacteria

FungiFungi

BacteriaBacteria

VirusesViruses

VirusesViruses

NucleusNucleus

In�ammasomeFormationIn�ammasomeFormation

LysosomeLysosome

MitochondriaMitochondria

EndoplasmicReticulumEndoplasmicReticulumEndoplasmicReticulumReticulumEndoplasmicReticulumEndoplasmicReticulumEndoplasmicReticulumEndoplasmicReticulumEndoplasmicReticulumEndoplasmicReticulumEndoplasmicReticulum


Innate Immune Signaling

biolegend.com

EndosomesEndosomes

Toll - Like Receptor LigandsToll - Like Receptor Ligands

TLR1 - Triacetylated lipopeptidesTLR2 - Glycolipids, lipopeptides, lipoproteinsTLR3 - Double-stranded RNATLR4 - Lipopolysaccharide, heat shock proteins, �brinogenTLR5 - FlagellinTLR6 - Diacyl lipopeptidesTLR7 - Single-stranded RNATLR8 - Single-stranded RNA (Human) Imidazoquinoline, poly T oligonucleotides (Mouse)

TLR9 - Unmethylated CpG DNA, haemozoinTLR10 - ? TLR11* - Flagellin, pro�linTLR12* - Pro�linTLR13* - 23s rRNA


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