Developing New Assays for FXM

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Developing New Assays for FXM
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• Need to monitor signal transduction

• Many unexpected phenotypes from RNAi perturbation
  experiments

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• Phosphoprotein assay
• IP3 Assay
• Translocation Assay
• Others

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• Phosphoprotein assay
• IP3 Assay
• Translocation Assay
• Others

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Phosphoprotein targets suggested by committee
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Phosphoprotein readout for FXM

• 2 modest responses
• Several weak responses

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Increased P-Akt response to C5a in Gai2 knockdown
cells
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P-Akt response is PTx sensitive
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Differential effect of PTx on Ca2 and PIP3 in
Gai2 knockdown cells
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Decreased P-p38 response to UDP in Gaq knockdown
cells
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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

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• Phosphoprotein assay
• IP3 Assay
• Translocation Assay
• Others

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IP3 Assay

• Gain insight on Ca2 responses to different
  ligands

• Dissect interactions between Ca2 and PIP3 modules

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IP3 Assay

• Competitive binding assay
• Direct measurement
• Low IP3, Ca2/Mg2 in buffer interferes with
  assay

• Accumulation assay
• Indirect measurementIP accumulation
• It works!

• PLCd-PH translocation assay
• Single cell in vivo assay
• Need development

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• Phosphoprotein assay
• IP3 Assay
• Translocation Assay
• Others

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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

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Translocation Assay

• Focus on XFP reporters
• most constructs are available
• use ligand cocktail to screen for response

• Adopt available FRET assays

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• Phosphoprotein assay
• IP3 Assay
• Translocation Assay
• Others

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Other Assays...

• Developed
• cAMP
• Lipid
• macropinocytosis

• To be developed
• tomorrows session

• We need robust assays!

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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
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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

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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