1. Developing New Assays for FXM Assay!!!

2. FXM, 4 th Annual AfCS Meeting UDP C5a IgG 2a Ca 2+ Akt-PH  Need to monitor signal transduction  Many unexpected phenotypes from RNAi perturbation experiments

3. FXM, 4 th Annual AfCS Meeting UDP C5a IgG 2a Ca 2+ Akt-PH  Phosphoprotein assay  IP3 Assay  Translocation Assay  Others

4. FXM, 4 th Annual AfCS Meeting UDP C5a IgG 2a Ca 2+ Akt-PH  Phosphoprotein assay  IP3 Assay  Translocation Assay  Others

5. FXM, 4 th Annual AfCS Meeting Phosphoprotein targets suggested by committee UDP C5a IgG 2a Ca 2+ Akt-PH Erkp38 p90 MARCKS Pyk2 paxillin p70 S6K CREB RiboS6 FKHR MLC cPLA2, RiboS6, CREB Rac PAK mTOR p70 S6K Pyk2, FAK paxillin GSK3 

6. FXM, 4 th Annual AfCS Meeting UDP C5aIgG 2a Ca 2+ Akt-PH Pyk2 mTOR p70 S6K Akt Erk p38 p90 Pyk2 p70 S6K Phosphoprotein readout for FXM  2 modest responses  Several weak responses

7. FXM, 4 th Annual AfCS Meeting Increased P-Akt response to C5a in G  i2 knockdown cells Akt (S473) RhoGDI NS C5a 1’ C5a 3’ C5a 10’ C5a 30’ NS C5a 1’ C5a 3’ C5a 10’ C5a 30’ NS control G  i2 (min)

8. FXM, 4 th Annual AfCS Meeting P-Akt response is PTx sensitive NS C5a 1’ C5a 3’ C5a 10’ UDP 3’ NS C5a 1’ C5a 3’ C5a 10’ UDP 3’ +PTx-PTx Akt (S473) control +PTx-PTx NS C5a 1’ C5a 3’ C5a 10’ UDP 3’ NS C5a 1’ C5a 3’ C5a 10’ UDP 3’ Akt (S473) G  i2 RAW 264.7 control C5aR G  i2 G  q Anti-G  i2 (min)

9. FXM, 4 th Annual AfCS Meeting Differential effect of PTx on Ca 2+ and PIP3 in G  i2 knockdown cells C5aR G  iG  PI3K PLC Ca 2+ PIP3 G  i2 knockdown PTx PIP3 C5aR G  iG  PI3K PLC Ca 2+ control PTx GqGq

10. FXM, 4 th Annual AfCS Meeting Decreased P-p38 response to UDP in G  q knockdown cells p38MAPK (T180/Y182) Pyk2 (Y402) RhoGDI NS UDP 1’ UDP 3’ UDP 10’ NS UDP 1’ UDP 3’ UDP 10’ NS control GqGq (min) RAW 264.7 control C5aR G  i2 G  q Anti-G  q

11. FXM, 4 th Annual AfCS Meeting Phosphoprotein assay  Continue screening for new phospho- specific antibodies  Develop new technologies: SILAC, AQUA peptide, Bio-Plex  Examine phenotypes in RNAi knockdown cells with FXM P-Ab mixes 1 set of samples for 21 lines replicated samples for 6 lines

12. FXM, 4 th Annual AfCS Meeting UDP C5a IgG 2a Ca 2+ Akt-PH  Phosphoprotein assay  IP3 Assay  Translocation Assay  Others

13. FXM, 4 th Annual AfCS Meeting UDP C5a IgG 2a Ca 2+ Akt-PH Akt p38 IP3 PLC IP3PI(4,5)P2PI(3,4,5)P3 PI3K IP3R Ca 2+ + +

14. FXM, 4 th Annual AfCS Meeting IP3 Assay UDP IgG 2a + anti-IgG C5a Ligand Macro. Bio. lab  Gain insight on Ca 2+ responses to different ligands  Dissect interactions between Ca 2+ and PIP3 modules

15. FXM, 4 th Annual AfCS Meeting IP3 Assay  Competitive binding assay Direct measurement Low [IP3], Ca 2+ /Mg 2+ in buffer interferes with assay  PLC  -PH translocation assay Single cell in vivo assay Need development  Accumulation assay Indirect measurement—IP accumulation It works!

16. FXM, 4 th Annual AfCS Meeting UDP C5a IgG 2a Ca 2+ Akt-PH  Phosphoprotein assay  IP3 Assay  Translocation Assay  Others

17. FXM, 4 th Annual AfCS Meeting Translocation Assay  GPCR activation and desensitization Arrestin Grk2  Cytoskeleton Actin coronin  Rac/Cdc42 PAK-PBD  PI3K/PIP3 p85 Akt-PH Btk Ship FoxO3  Calcium/PKC PKC CaM-M13 CaMKII Erk NFAT NF-  B  Tyrosine kinase Syk  PLC/IP3 PLC  -PH

18. FXM, 4 th Annual AfCS Meeting Translocation Assay  Focus on XFP reporters: most constructs are available use ligand cocktail to screen for response  Adopt available FRET assays:

19. FXM, 4 th Annual AfCS Meeting UDP C5a IgG 2a Ca 2+ Akt-PH  Phosphoprotein assay  IP3 Assay  Translocation Assay  Others

20. FXM, 4 th Annual AfCS Meeting Other Assays...  Developed: cAMP Lipid macropinocytosis  To be developed: tomorrow’s session  We need robust assays!

21. FXM, 4 th Annual AfCS Meeting How Many Assays Do We need  Estimate based on studies in yeast (Michnick report; G. Yao et al, PLoS Biol. 2 (3), p355-367, March 2004) ~10-20 interacting proteins per signaling step ~200 proteins on FXM parts list We need 10-20 assays on non-randomly selected targets

22. FXM, 4 th Annual AfCS Meeting How Many Assays Do We need  FXM network: We need ~10 assays, one for each signaling step GPCR G  G  PLC Fc  R PTK PI3K PIP3 IP3 IP3R Ca 2+ Number of assays needed will not increase proportionally as we expand beyond FXM

23. FXM, 4 th Annual AfCS Meeting Acknowledgement Phosphoprotein assay Yan Ni Audra Wendt Cell Preparation Lab Macrophage Biology Lab Antibody Lab IP3 assay Dianne DeCamp Meghdad Rahdar Cell Preparation Lab Translocation assay Microscopy Lab Molecular Biology Lab