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A Single Cell PIP 3 Assay Based on Akt PH domain Translocation Nancy O’Rourke Microscopy Lab, Stanford University

A Single Cell PIP 3 Assay Based on Akt PH domain Translocation Nancy O’Rourke Microscopy Lab, Stanford University Jim Whalen, Takako Mukai, Wei Sun Park, Liz Gehrig Grischa Chandy, Tobias Meyer. Macrophage Lab, UCSF Tamara Roach Robert Rebres. Bioinformatics Lab, UCSD Ilango Vadivelu

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A Single Cell PIP 3 Assay Based on Akt PH domain Translocation Nancy O’Rourke Microscopy Lab, Stanford University

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  1. A Single Cell PIP3 Assay Based on Akt PH domain Translocation Nancy O’Rourke Microscopy Lab, Stanford University Jim Whalen, Takako Mukai, Wei Sun Park, Liz Gehrig Grischa Chandy, Tobias Meyer

  2. Macrophage Lab, UCSF Tamara Roach Robert Rebres Bioinformatics Lab, UCSD Ilango Vadivelu Bob Sinkovits Molecular Biology Lab, Caltech Iain Fraser Joelle Zavzavadjian Leah Santat Jamie Liu Estelle Wall Kavitha Dhandapani

  3. A Single Cell PIP3 Assay Based on Akt PH domain Translocation Assay Protocol and Analysis Testing the FXM Ligands Perturbation of the System with RNAi and Chemical Reagents

  4. Assay Protocol Transfect YFP Akt PH domain and CFP KRAS CAAX Cells in 8-well chamber slide Serum Deprive 1 hour Run at 37o Image on confocal microscope 40 scans w/ 10 second interval Ligand added at 50 seconds

  5. Image Analysis Time 0 secs. 140 secs. Correlation Coefficient -0.3238 0.4148 C5a

  6. Akt PH Domain Translocation after 100 nM C5a Stimulation Individual and Average Responses

  7. Diverse Patterns of Akt PH Domain Translocation

  8. Responses to C5a, UTP and FcgR1 C5a responses are consistent and robust. C5a stimulation used for current perturbation studies. Translocation does not occur following UDP/UTP stimulation. Responses to FcgR1stimulation vary with cell batch.

  9. Akt PH Domain Translocation with UTP, FcgR1 and C5a Stimulation 100 mM UTP 0.44 mM F(ab’)2 100 nM C5a

  10. Variability in the FcgR1 Response

  11. Perturbation Experiments C5a stimulation utilized for perturbation studies. Lentiviral cell lines expressing GFP interfere with the YFP-Akt PH fluorescence signal. Three CD4 lentiviral cell lines: SHIP1, Gb4 and Gai3. Chemical perturbation studies initiated with LY

  12. Knockdown of SHIP1 Increases Translocation

  13. Gi3 SHIP1 UCIP 188 98 IB: SHIP1 Testing of lentiviral lines for knockdownefficacy SHIP1 UCIP Gi3 49 38 IB: G alpha i3 99% Knockdown in UC lines. Caltech Lab

  14. Knockdown of Gb4 does not alter Translocation

  15. Results are Consistent with Population Ca2+ Results UCSF lab

  16. LY Pretreatment Decreases the Translocation 10 minute pretreatment with100 mM LY

  17. Syk Lyn, Hck GRK2 (4,5,6) P2Y2,6 C5aR CD64 Arrestin 2,3 PI3K p85a, p110a,d Gai2,3 Gaq, Ga11 PTEN Gb1,2 (4,5) SHIP-2 (1) p101, p110g PLCb2,3 (4) PLCg1,2 PI(3,4,5)P3 Btk, Tec Akt1,2,3 IP3KTB(C) IP3R1,2,3 Lentiviral Lines with CD4 marker Gil Sambrano Lily Jiang LY PMCA1,3 (4) SERCA2,3

  18. FXM Translocation Data Display

  19. Treatment Summary N - 8 cells % cells with Translocation – 88%

  20. Conclusions The Akt PH domain translocation assay can be employed to probe PIP3 signaling pathway C5a provides a consistent and robust response in the translocation assay Results from preliminary studies confirm the roles of PI3K and SHIP1 in PIP3 signaling. FXM Translocation Display Available

  21. Future Directions Increase throughput of assay. Screen Additional Non-fluorescent Lentiviral Cell Lines Develop Methods for Using Duplex RNAi with Translocation Assay Refine FXM Display

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