1. Antibody Lab B Cell Ligand Screen Quantify ligand-induced changes in site-specific phosphorylation of selected signaling proteins Erica Turon, technician and Heping Han, Lead Scientist

2. Antibody Lab B Cell Ligand Screen Aim: sample diversity of responses to inputs Approach: Probe Western blots with mixture of site specific, phosphorylation sensitive antibodies Goal: resolve and quantify 5-7 phosphoproteins per lane of gel Progress: 2 mixtures of antibodies for 11 phosphoproteins

3. Targets of Current Mixes of Phosphospecific Antibodies Mix 1 Stat 6: Y641 p90RSK: S381 Akt: S473 ERK 1 T202/Y204 ERK 2 T183/Y185 Mix 4 PKC  S916 Stat 3 Y705 NF  B p65 S536 JNK long T183/Y185 JNK short T183/Y185 p38 MAPK T180/Y182 Criteria: single band of appropriate size, ligand sensitive Tested 64 different antibody preparations (1 out of 7) All antibodies from Cell Signaling Technology, except one

4. B Cell Receptor Map * *

5. Untreated Anti-IgM IL-4 Pos.Cont.A Anti-IgM = 0.3  M, IL-4 = 0.4 nM, Positive control A = Anti-IgM + IL-4 All treatment was 5 min Phosphospecific Antibody Mix 1 p90RSK ERKs Stat6 Akt

6. Anti-IgM (0.3  M ) IL-4 (0.34 nM) Anti-CD40 (65 nM) 2.5’ 5’ 15’ 30’ + Control Untreated <p90RSK <Akt > ERKs <Stat6 <G  Ligand Screen Time Courses EWC020314B_EA Cell ID = EWC020319D Anti-IgM = 0.3  M; IL-4 = 0.34 nM ; CD-40 = 65 nM

7. Imaging No film or radioactivity ECL Plus (has fluorescent by- product) Imager detects fluorescence ImageQuant software to save image Biren Zhao with Storm 860 Imager from Molecular Dynamics

8. Quantification Nick Wong: searched for better software, implemented ImageMaster: Automatically finds lanes, semi-automatic for bands

10. Interleukin 4 Phosphorylation of Stat 6 50 fold increase in phosphorylation of Stat6 Single scale: Cannot see changes in phosphorylation of other proteins

11. Interleukin 4 Two y axes scaled differently Reveals phosphorylation of Akt (fuscia)

12. Additional Ligands: Gi-mediated Responses Sphingosine 1-phosphate and Chemokines: SDF, BLC, ELC, SLC Increased phosphorylation of Akt, ERKs and p90RSK with  max. response at first time point  More rapid initial decline of ERKs Time course pattern similar to B cell receptor Gq, Gs pathways: no effect of terbutaline, PGE2, LPA, 2-MA

13. Antibody Mix 1 Additional Ligands with No Response IL-10 Interferon  IGF-1 TNF  Leukotriene B4 fMLP Neurokinin B Responses with Antibody Mix 4?

14. Positive Control B = 0.34 nM IL-10 and 65 nM anti-CD40 treated B cells for 5 min. EA239_April 1002 Anti-CD40 + IL-10 0’ 5’ 15’ Pos. Cont. B Phosphospecific Antibody Mix 4 p38MAPK NF  B p65 PKC  Stat3 JNK

15. CD40 Signaling Map ** *

16. Phosphoprotein Antibody Mix 4 Responses to Anti-CD40 Early response: phosphorylation of NF  B p65 JNKs: delayed response (similar to ERKs and Akt) separate y axis

17. Phosphoprotein Antibody Mix 4 Responses to IL-4 and Anti-IgM IL-4: Rate of Stat 3 phosphorylation slower than Stat 6 Anti-IgM: modest responses

18. Phosphoprotein Antibody Mix 4 Ligand Screen Anti-IgM, Anti-CD40 and IL-4 quantified n=2 for data shown Data on website soon Becky Fulin, Technician

19. Our Group Antibody Lab: Lead scientist: Heping Han Technicians: Eduardo Arteaga, Becky Fulin, Nick Wong, Biren Zhao Dallas Labs: Data Manager: Lonnie Sorrells Administrator: Kim Edwards

20. Future Goals Ligand Screens B cell WEHI 231 Myocytes Multi-ligand Luminex fluorescent bead technology Determine concentration of signaling proteins Focus on PIP3 Modules in WEHI 231 cells Characterize/utilize phosphospecific antibodies Collaboration with Cell Signaling Technology Eduardo Arteaga