Functional Responses in the Additional Cells

Cell Preparation and Analysis Laboratory and Antibody

Laboratory (Dianne DeCamp, Heping Han, Robert Hsueh,

Keng-Mean Lin, Yan Ni)

The Players

RAW 264.7 J774A.1 IC-21 AtT-20 3T3L1 N1E-115 Neuro2A

lineage macrophage macrophage macrophage pituitary fibroblast neuoblast neuroblastestablished culture conditions

Y Y Y Y Y Y N

total protein (µg/ 106) 130 115 200 400

total RNA (µg/ 106) 15 15 30 30 50transfectioninfectionRNAiResponses

Ligand Receptor(s) cAMP Calcium

5'-(N-Ethylcarboxamido)adenosine Adenosine A2B

Calcitonin-gene-related peptideFenoldopam Hydrobromide D1Isoproterenol Adrenergic 1 Melanocyte Stimulating Hormone MC5Prostaglandin E2Terbutaline Adrenergic 2B-lymphocyte chemoattractant (BLC) CXCR5C5a C5a receptor

(CD88)

CX3C Chemokine CX3CR1 (Fractalkine Receptor)

CXCL11/Interferon-inducible T-cell alpha chemoattractant(I-TAC)

CXCR3

DopamineELC CCR7Estradiol Estrogen receptor-Neurokinin NK2(Tachykinin2)Leukotriene D4 (LTD4) CysLT1Lysophatidic Acid (LPA) Edg-2,4,7Platelet-Activating Factor (PAF) PAF receptorSDF CXCR4Serotonin 5HT 1B &2C(1A?)Uridine 5'-Triphosphate (UTP) P2Y4

Vasopressin V1

Test Ligands

Protocols for Calcium Assays in RAW264.7 and J774 Cells

RAW264.7 J774

Detachment using PBS/EDTA

Loading with Fluo3 AM (3 M)

Recovery

Washing

Adjusting the cell density to 1 x106 cells/ml

stimulate with different ligands

Adjusting the cell density to 1 x106 cells/ml

Note: Cells are in suspension throughout the calcium assay.

Time (sec)

J774A.1RAW 264.7

C5a (M)[C

a2+

] (n

M)

400

200

[Ca

2+]

(nM

)

300

250

200

Platelet-ActivatingFactor (M)

[Ca

2+]

(nM

)

200

400

[Ca

2+]

(nM

) 300

250

200

Lyso-phosphatidic Acid (M) [C

a2+

] (n

M)

200

400

[Ca

2+]

(nM

) 300

250

200

100 200 3000 100 200 3000

Changes in Intracelluar Calcium

Time (sec)

Protocols for cAMP Assays in RAW264.7 Cells

RAW264.7

Plate cells overnight in the 96-well plate at 5 x105 cells/ml

stimulate with different ligands

Stop the reaction by addition of 100% ethanol at different time points

Process cell lysate

Assay with cAMP Biotrak EIA cAMP Biotrak EIA kit

Note: the treatment is performed in the absence of any phosphodiesterase inhibitors.

cAMP Responses in RAW264.7 Cells

Time (min)

[ISO] = 1 M[TER] = 10 M[PGE] = 50 M

cAMP Responses in RAW 264.7

Time (min)

Fol

d

N=3

Test LigandsLigand Receptor(s) cAMP Calcium

5'-(N-Ethylcarboxamido)adenosine Adenosine A2B

Calcitonin-gene-related peptideFenoldopam Hydrobromide D1Isoproterenol Adrenergic 1 Y (n=2) Melanocyte Stimulating Hormone MC5Prostaglandin E2 Y (n=2)Terbutaline Adrenergic 2 Y (n=2)B-lymphocyte chemoattractant (BLC) CXCR5 NR (n=1)C5a C5a receptor

(CD88)Y (n=3)

CX3C Chemokine CX3CR1 (Fractalkine Receptor)

CXCL11/Interferon-inducible T-cell alpha chemoattractant(I-TAC)

CXCR3

Dopamine NR (n=1)ELC CCR7 Y (n=1)Estradiol Estrogen receptor-Neurokinin NK2(Tachykinin2) NR (n=1)Leukotriene D4 (LTD4) CysLT1Lysophatidic Acid (LPA) Edg-2,4,7 Y (n=3)Platelet-Activating Factor (PAF) PAF receptor Y (n=3)SDF CXCR4 NR (n=1)Serotonin 5HT 1B &2C(1A?) NR (n=1)Uridine 5'-Triphosphate (UTP) P2Y4

Vasopressin V1 NR (n=1)

Summary/Immediate Future

• Initial culture conditions are established for the cells

• Previously established protocols and assays are being applied to studies of the additional cell lines.

• cAMP and Calcium assays are functional, but can be modified.

• Preliminary ligand response testing continues

LipopolysaccharidesCpG DNA Poly (I:c)

Imiquimod. R848Lipid proteins

mannans

Oxidized EPA

Thr2Thr6

Thr9 Thr3 Thr7Thr10

(LPS)

T cells

Cell Mediated Immunity

Influenceadaptive Immuneresponse

Apoptosis of host cells

Direct antimicrobial response

Bacterialdeath

Oxidized EPA

Toll-like Receptor Pathways

Multiplex Western Blot with Phosphospecific Antibody Mix 2 RAW 264.7 Cells +/-LPS or IL-4

< P-STAT3

< P-NFkB p65

>

<P-IKB

< P-PKC

P-JNK

<P-p38MAPK

LPS = 10 g/ml, IL-4 = 0.34 nM

Antibody Mix 2

RAW one Hour

1% serum

RAW 24 Hour 1% serum

WEHI control

0 LPS IL4 0 LPS IL4 + -

Phospho-NFk B p65

0

1

2

3

4

untreated LPS IL-4

Ligand (10 min)

Fo

ld C

han

ge

Phospho-JNK(long)

0

5

10

15

20

25

30

untreated LPS IL-4

Ligand (10 min)

Fo

ld C

han

ge

Phospho-p38 MAPK

0

10

20

30

40

50

untreated LPS IL-4

Ligand (10 min)F

old

Ch

ang

e

IL-4 Pathway

< P-STAT6

> P-ERKs

< P-Akt

< P-p90RSK

Multiplex Western Blot with Phosphospecific Antibody Mix 1 RAW 264.7 Cells +/-LPS or IL-4

Antibody Mix 1

RAW one Hour

1% serum

RAW 24 Hour 1% serum

WEHI control

0 LPS IL4 0 LPS IL4 + -

LPS = 10 g/ml, IL-4 = 0.34 nM

Phospho-STAT6

0

5

10

15

20

25

30

untreated LPS IL-4

Ligand (10 min)

Fo

ld C

ha

ng

e

Phospho-ERK1

02468

10

untreated LPS IL-4

Ligand (10 min)

Fo

ld C

han

ge

Phospho-p90RSK

02468

10

untreated LPS IL-4

Ligand (10 min)

Fo

ld C

han

ge

24-hour Serum Deprivation Increases Ligand-Stimulated Protein Phosphorylation in RAW 264.7 Cells

Similar Increases for: • P-STAT6 • P-JNK (long)• P-JNK (short)• P-ERK1• P-ERK2• P-p70S6K• P-Ribosomal S6

Not true for WEHI-231

Small or no Difference: P-Akt, P-NFB p65, PKC

Phospho-p38 MAPK

0

10

20

30

40

50

untreated LPS I04 untreated LPS I04

Ligand (10 min)

Fo

ld C

ha

ng

e

1hr. 1%serum 24hr. 1%serum

Phospho-p90 RSK

0

2

4

6

8

10

untreated LPS IL-4 untreated LPS IL-4

Ligand (10 min)

Fo

ld C

han

ge

1 hr. 1%Serum 24 hr. 1%Serum

Data from Protein and Cell LabsWestern Blots of Bulk Protein Phosphorylation in RAW 264.7

< 111 < 80 < 61< 48

< 36

< 25

< 19< 14< 6

Phospho-Thr Antibody Phospho-Tyr Antibody

Calyculin A (nM) Sodium pervanadate (M)

0 5 10 20 40 0 5 50 100 200 MW

Phospho-threonine and/or phospho-tyrosine antibodies may be useful for future IP and phosphoprotein identification in RAW cells

____ ____ ____ _____ 60 min 30 min

• Phosphospecific antibody Mixes 1 and 2 are ready candidates for monitoring protein phosphorylation in RAW 264.7 cells • The same is likely true

• for or other hematopoietic cells (J774)• for AtT20 (neruorendocrine) cells based on basal phosphorylation patterns (not shown)

Future

The Usual SuspectsCell Preparation and Analysis Laboratory

Julie Collins

Richard Davis

Joella Grossoehme

Christine Horvath

Jason Polasek

Meghdad Rahdar

Debalina Siddeeq

Megan Smith

Melissa Stalder

Antibody Laboratory

Eduardo Arteaga

Becky Fulin

Ila Oxendine

Greta Sartoni-D’Ambrosia

Nick Wong

Biren Zhao