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Developing New Assays for FXM

Assay!!!. Developing New Assays for FXM. IgG 2a. UDP. C5a. Akt-PH. Ca 2+. Need to monitor signal transduction. Many unexpected phenotypes from RNAi perturbation experiments. IgG 2a. UDP. C5a. Akt-PH. Ca 2+. Phosphoprotein assay IP3 Assay Translocation Assay Others. IgG 2a. UDP.

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Developing New Assays for FXM

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  1. Assay!!! Developing New Assays for FXM

  2. IgG2a UDP C5a Akt-PH Ca2+ • Need to monitor signal transduction • Many unexpected phenotypes from RNAi perturbation experiments

  3. IgG2a UDP C5a Akt-PH Ca2+ • Phosphoprotein assay • IP3 Assay • Translocation Assay • Others

  4. IgG2a UDP C5a Akt-PH Ca2+ • Phosphoprotein assay • IP3 Assay • Translocation Assay • Others

  5. IgG2a UDP C5a paxillin Pyk2, FAK Rac PAK p70 S6K FKHR Akt-PH MARCKS Erk p38 RiboS6 mTOR GSK3a/b p90 p70 S6K CREB MLC Pyk2 Ca2+ cPLA2, RiboS6, CREB paxillin Phosphoprotein targets suggested by committee

  6. C5a IgG2a UDP Pyk2 p70 S6K Akt Akt-PH Erk p38 mTOR p90 p70 S6K Pyk2 Ca2+ Phosphoprotein readout for FXM • 2 modest responses • Several weak responses

  7. NS C5a 1’ C5a 3’ C5a 10’ C5a 30’ NS NS C5a 1’ C5a 3’ C5a 10’ C5a 30’ NS Gai2 control Akt (S473) RhoGDI (min) (min) Increased P-Akt response to C5a in Gai2 knockdown cells

  8. NS NS C5a 1’ C5a 3’ C5a 10’ UDP 3’ NS C5a 1’ C5a 3’ C5a 10’ UDP 3’ +PTx -PTx control Akt (S473) NS NS C5a 1’ C5a 3’ C5a 10’ UDP 3’ NS NS C5a 1’ C5a 3’ C5a 10’ UDP 3’ Akt (S473) Gai2 RAW 264.7 control C5aR Gai2 Gaq +PTx -PTx Anti-Gai2 (min) P-Akt response is PTx sensitive

  9. control Gai2 knockdown C5aR C5aR PTx PTx GaiGbg Gaq GaiGbg PI3K PI3K PLC PLC PIP3 PIP3 Ca2+ Ca2+ Differential effect of PTx on Ca2+ and PIP3 in Gai2 knockdown cells

  10. NS UDP 1’ UDP 3’ UDP 10’ NS NS UDP 1’ UDP 3’ UDP 10’ NS Gaq control p38MAPK (T180/Y182) Pyk2 (Y402) RhoGDI RAW 264.7 control C5aR Gai2 Gaq Anti-Gaq (min) Decreased P-p38 response to UDP in Gaq knockdown cells

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

  12. IgG2a UDP C5a Akt-PH Ca2+ • Phosphoprotein assay • IP3 Assay • Translocation Assay • Others

  13. IgG2a UDP C5a Akt Akt-PH p38 PLC PI3K IP3 + + IP3 PI(4,5)P2 PI(3,4,5)P3 IP3R Ca2+ Ca2+

  14. Macro. Bio. lab UDP C5a IgG2a+ anti-IgG Ligand IP3 Assay • Gain insight on Ca2+ responses to different ligands • Dissect interactions between Ca2+ and PIP3 modules

  15. IP3 Assay • Competitive binding assay • Direct measurement • Low [IP3], Ca2+/Mg2+ in buffer interferes with assay • Accumulation assay • Indirect measurement—IP accumulation • It works! • PLCd-PH translocation assay • Single cell in vivo assay • Need development

  16. IgG2a UDP C5a Akt-PH Ca2+ • Phosphoprotein assay • IP3 Assay • Translocation Assay • Others

  17. Translocation Assay • GPCR activation and desensitization • Arrestin • Grk2 • PI3K/PIP3 • p85 • Akt-PH • Btk • Ship • FoxO3 • Calcium/PKC • PKC • CaM-M13 • CaMKII • Erk • NFAT • NF-kB • Tyrosine kinase • Syk • Rac/Cdc42 • PAK-PBD • Cytoskeleton • Actin • coronin • PLC/IP3 • PLCd-PH

  18. Translocation Assay • Focus on XFP reporters: • most constructs are available • use ligand cocktail to screen for response • Adopt available FRET assays:

  19. IgG2a UDP C5a Akt-PH Ca2+ • Phosphoprotein assay • IP3 Assay • Translocation Assay • Others

  20. Other Assays... • Developed: • cAMP • Lipid • macropinocytosis • To be developed: • tomorrow’s session • We need robust assays!

  21. 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. GPCR FcgR GaGbg PTK PLC PI3K IP3 IP3R PIP3 Ca2+ How Many Assays Do We need • FXM network: • We need ~10 assays, one for each signaling step • Number of assays needed will not increase proportionally as we expand beyond FXM

  23. 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

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