(—THIS SIDEBAR DOES NOT PRINT—)

D E S I G N G U I D E

This PowerPoint template produces an 84”x36” presentation poster. You can use it to create your research poster and save time placing titles, subtitles, text, and graphics. When you are ready to print your poster, go online to megaprint.com or any service bureau of your choosing.

Need assistance? Contact the marketing group. David.scher@cellsignal.com

Q U I C K S TA R T

Zoom in and out As you work on your poster zoom in and out to the level that is more comfortable to you. Go to VIEW > ZOOM.

Title, Authors, and Affiliations

Start designing your poster by adding the title, the names of the authors, and the affiliated institutions. You can type or paste text into the provided boxes. The template will automatically adjust the size of your text to fit the title box. You can manually override this feature and change the size of your text.

T I P : The font size of your title should be bigger than your name(s) and institution name(s).

Adding Logos / Seals

We have already inserted the CST logo in position. You can insert a logo for additional presenters institution by dragging and dropping it from your desktop, copy and paste or by going to INSERT > PICTURES. Logos taken from web sites are likely to be low quality when printed. Zoom it at 100% to see what the logo will look like on the final poster and make any necessary adjustments.

Photographs / Graphics

You can add images by dragging and dropping from your desktop, copy and paste, or by going to INSERT > PICTURES. Resize images proportionally by holding down the SHIFT key and dragging one of the corner handles. For a professional-looking poster, do not distort your images by enlarging them disproportionally.

Image Quality Check Zoom in and look at your images at 100% magnification. If they look good they will print well. If they do not look like they are a high enough quality, contact the marketing group for support. ataylor@cellsignal.com

Good

prin

tingqu

ality

Badprintin

gqu

ality

ORIGINAL DISTORTED

Q U I C K S TA RT ( c o n t . )

How to change the template theme

This document has been constructed to keep the characteristics of the CST brand. Templates are available or can be custom prepared for additional dimensions, depending on the requirements of the conference you will be attending.

How to add Text The template comes with a number of pre-formatted placeholders for headers and text blocks. You can add more blocks by copying and pasting the existing ones or by adding a text box from the HOME menu.

Text size and Fonts

Adjust the size of your text based on how much content you have to present. The default template text offers a good starting point. Follow the conference requirements, and try to keep in mind that the posters text should remain comfortably legible from viewing distances of 6’ – 10’. The fonts chosen for this template reflect the fonts in our corporate brand and are also chosen for their legibility and universal availability. DO NOT use any other fonts without approvval from the Marketing Group.

How to add Tables

To add a table from scratch go to the INSERT menu and click on TABLE. A drop-down box will help you select rows and columns.

You can also copy and a paste a table from Word or another PowerPoint document. A pasted table may need to be re-formatted by RIGHT-CLICK > FORMAT SHAPE, TEXT BOX, Margins.

Graphs / Charts You can simply copy and paste charts and graphs from Excel or Word. Some reformatting may be required depending on how the original document has been created.

How to change the column configuration

RIGHT-CLICK on the poster background and select LAYOUT to see the column options available for this template. The poster columns can also be customized on the Master. VIEW > MASTER.

How to remove the info bars

If you are working in PowerPoint for Windows and have finished your poster, save as PDF and the bars will not be included. You can also delete them by going to VIEW > MASTER. On the Mac adjust the Page-Setup to match the Page-Setup in PowerPoint before you create a PDF. You can also delete them from the Slide Master.

Save your work Save your template as a PowerPoint document. For printing, save as PowerPoint or “Print-quality” PDF.

Poly-ADP-ribose polymerases (PARPs) catalyze the transfer of ADP-ribose from β-NAD+ and release nicotinamide in the process. ADP-ribosylation predominantly occurs on amino acid side chains of proteins (such as lysine, arginine, glutamate, aspartate, cysteine, serine), but it has also been described to occur on protein amino termini as well as on DNA and tRNA.1 The most widely studied PARPs (PARP1, 2, 5a and 5b) can synthesize linear or branched chains of up to ~200 ADP-ribose units (PARylation).2 However, there are 13 additional PARPs which transfer only a single ADP-ribose unit to their target residue (MARylation). The best-known function of poly-ADP-ribose chains is to serve as a scaffold for the recruitment of DNA repair proteins that contain PAR-binding modules to sites of DNA damage. ADP-ribosylation is also involved in a variety of additional cellular processes, including cell stress responses, mitotic spindle formation, chromatin decondensation, retroviral silencing, RNA biology, and transcription.3 Even though MAR/PARylation is of central importance to cellular function, there are no commercially available antibodies that recognize both MARylated and PARylated proteins. Therefore, novel rabbit monoclonal antibodies have been produced and characterized against this modification on proteins, and their utility for the detection of ADP-ribosylated proteins by ELISA, western blot, dot blot, and immunofluorescence assays has been validated.

INTRODUCTION

REFERENCES 1.  Koch-Nolte, F. et al. (2008) Front Biosci 13, 6716-29.

2.  Leung, A.K. (2014) J Cell Biol 205, 613-9.

3.  Gupte, R. et al. (2017) Genes Dev 31, 101-126.

4.  https://www.cellsignal.com/contents/resources/protocols/ resources-protocols

5.  Jones, P. et al. (2009) J. Med. Chem. 2009, 7170–7185.

Antibody Development

METHODS Polyclonal antibodies were produced by modifying lysine residues on KLH using periodate chemistry. Rabbits were selected for monoclonal antibody development based on reactivity in ELISA and western blot assays. Rabbit monoclonal clones were then produced, tested, and selected for scale-up and additional testing. ELISA, Western blot, and immunofluorescence assays were performed as described.4 Hydrogen peroxide treatment was performed at 500mM for 5 min. PDE1 treatment was performed at 0.5mg/mL for 4hr at 37°C. PARG treatment was performed at 5mM for 4hr at 37°C. The immunofluorescence PARylation assay was performed as described by Jones et al. and cells were imaged on a ImageXpress Micro XLS and quantified using MetaXpress.5

CONCLUSIONS Four antibody clones were isolated and characterized which showed distinct banding patterns and levels of induction of cellular ADP-ribosylation on Western blots of cells stimulated with PARylation-activating treatments such as hydrogen peroxide. Clone E6F6A which was selected for commercial release was validated for use in Western blot, dot blot, ELISA, and immunofluorescence assays. Initial studies also show that the antibody is a useful tool for the enrichment of MAR/PARylated proteins or peptides prior to LC-MS/MS analysis. Further work is aimed at the understanding of the specificity of the four antibody clones. Development of new tools to study this critical post-translational modification should facilitate new discoveries of MARylated/PARylated proteins and their function during growth, development, and in disease settings.

Development Scheme

Figure 5: MAR/PAR antibodies detect mono-ADP-ribosylated PARP16. Lysates from HeLa PARP16 -/- cells left untransfected (UT) or transfected with wild-type SBP-tagged PARP16 (WT) or catalytically dead SBP-tagged PARP16 (CD). (*) ADP-ribosylated PARP16

Antibody Characterization

Figure 3: MAR/PAR antibodies detect both mono- and poly-ADP-ribosylated recombinant proteins by dot blot. The four clones show different levels of sensitivity toward modified proteins and do not bind to unmodified proteins.

Novel antibody reagents for the characterization of protein ADP-ribosylation Alvin Lu1, Rami Najjar2, Mario Niepel1, Matthew P. Stokes2

1Ribon Therapeutics, Lexington MA 02421 2Cell Signaling Technology, Inc., Danvers MA 01923

Alvin Lu email: alu@ribontx.com

www.cellsignal.com/posters

For Research Use Only. Not For Use in Diagnostic Procedures.©2018 Cell Signaling Technology, Inc. Cell Signaling Technology, and CST are trademarks of Cell Signaling Technology, Inc. All other trademarks are the property of their respective owners.

Samples PARP1

ADPr-PARP1 PARP3

ADPr-PARP3 CT

ADPr-CT Histone H3

ADPr-H3 BSA-peptide

ADPr-BSA-peptide BC-peptide

ADPr-BC-peptide

Confidential 1

Protein stain Anti-rabbit secondary

D3D4JBF D4F7PBF D9P7ZBF E6F6ABF D3D4JBF D4F7PBF D9P7ZBF E6F6ABF

MAR/PAR antibodies detect ADP-ribosylated recombinant proteins on dot blot. The four clones show different levels of sensitivity toward modified proteins but not unmodified version.

Confidential 2

D3D

4 JBF

D4 F 7P

BF

D9P

7 Z BF

E6 F 6A

BF

0 .0

0 .5

1 .0

No

rma

liz

ed

ra

tio

+ b u f fe r

+M a c ro D 2

+TARG 1

+S V P

Hydrolases Reported activity

MacroD2 Cleaves MAR

TARG1 Cleaves MAR/PAR

SVP Cleaves MAR/PAR

MAR/PAR antibodies show reduced signal against ADP-ribosylated PARP3 after incubation with hydrolases. Dot blot signal was normalized against buffer control.

Figure 4: A. MAR/PAR antibody signal is abrogated by treatment of COLO 205 cell lysates with PDE1 or PARG. B. MAR/PAR antibodies show reduced signal against ADP-ribosylated PARP3 after incubation with hydrolases. Dot blot signal was normalized against buffer control.

Figure 7: MAR/PARantibody(E6F6A)detectsdose-dependentinhibitionofPARylationbytalazoparibandniraparibinHelatreatedwithhydrogenperoxide.

0

2000

4000

6000

8000

10000

12000

D3D4J D9P7Z D4F7P E6F6A

BSA

BSA-MAR

H2O2 –+

D3D4J D9P7Z D4F7P E6F6A

–+ –+ –+

62

§ Polyclonal Stage

§ Rabbits immunized w/modified MAR-KLH

§ Test on HeLa –/+ H2O2

–+H2O2

Poly4Poly3Poly2Poly1

–+ –+ –+

Figure 1: MAR/PAR antibody development. A. Antibody development scheme. Rabbits were injected with ADP-ribose modified KLH, tested, and reactive animals selected for rabbit monoclonal antibody development. Clones were validated and selected for production of final reagent. B. Western blot testing of ADP-ribosylation polyclonal antibodies against HeLa cells untreated or treated with H2O2. Red box indicates polyclonal selected for rabbit monoclonal development.

KLH-Lysine-ADPribose

Polyclonalantibodies

Testing/Rabbitselection

Rabbitmonoclonalantibody(RmAb)development

RmAbclonetesting/selection

Figure 2: MAR/PAR rabbit monoclonal antibody validation. A. ELISA test of RmAb clones against BSA and ADP-ribosylated BSA. B. Western blot testing of ADP-ribosylation rabbit monoclonal antibodies against HeLa cells untreated or treated with H2O2.

Polyclonal Testing

Rabbit Monoclonal Testing

A.

B.

A. B.

+++H2O2

–+–––+

PDE1PARG

#83732(E6F6A)

A. B.

Dot Blot Testing

MAR/PAR Cleavage

Figure 6: Immunofluorescence staining of Hela cells stimulated with hydrogen peroxide and treated without and with Niraparib.

#83732(E6F6A)

PARP16 Over-Expression Immunofluorescence

Compound Screening Confidential 3

Anti-PARP16

D3D4JBF

D4F7PBF

D9P7ZBF

E6F6ABF

Anti-β-actin

10H

MAR/PAR antibodies detect ADP-ribosylation on over-expressed PARP16. Wild-type (WT) or catalytically dead mutant (CD) of SBP-tagged PARP16 was transiently expressed in HeLa PARP16 KO cells, with untransfected control (UT). ADP-ribosylated PARP16 is marked by (*), and total PARP16 is marked by (-).

* *

*

-

UT WT CD

0 .0 0 0 1 0 .0 0 1 0 .0 1 0 .1 10 .0

0 .5

1 .0

M A R /P A R IF A s s a y (C S T # E 6 F 6 A )

c o n c e n tra t io n (µM )

rela

tiv

e M

AR

/PA

R i

nte

ns

ity

T a la zo pa rib

N ira p a r ib