Figure 7. Functional assay of AGC-family substrates.

(A) Cell viability after siRNA transfection. H1703 cells were collected 60 hrs after transfection with the indicated siRNA, stained with Trypan Blue and counted. Numbers are mean ± SD from three different wells. Controls were transfected with scrambled siRNA. Results are representative of three independent experiments. (B) Immunoblotting of H1703 cells transfected with siRNAs. 60 hrs after transfection, cell lysates were made and probed with PARP antibody. (C) Immunoblotting of H1703 cells transfected with siRNAs. Cell lysates were made as in 7b and probed with PDGFRα antibody. (D) Immunoblotting of H1703 cells transfected with PDGFRα and SGTA siRNAs. 60 hrs after transfection, cell lysates were collected and probed with the antibodies indicated. (E) Immunoblotting of H1703 transfected with siRNAs. Cell lysates were made as in 7b and probed with the antibodies indicated. (F) Cell viability of H1703 cells transfected with siRNAs. siRNA transfected cells were collected and counted as in 7a. (G) Half life of PDGFRα in H1703 cells transfected with SGTA siRNA or treated with Gleevec. 48 hrs after siRNA transfection or overnight treatment with Gleevec (1 mM), cells were treated with the protein synthesis inhibitor cycloheximide (CHX, 0.1 mg/ml) for the indicated time and cell lysates were collected and probed with PDGFRα antibody. (H) SGTA S305 phosphorylation is required for SGTA/PDGFRα interaction. Cells were transfected with Flag-tagged wildtype SGTA (WT) or S305A mutant (SA). 32 hrs after transfection, cells were treated with Gleevec (1 uM) for 2.5 hrs and cell lysates were immunoprecipitated with Flag (left panel) or PDGFRα (right panel) and probed with antibodies indicated. (I) SGTA Ser³⁰⁵ is required for PDGFRα stability. SGTA siRNA knockdown cells were complemented with murine SGTA WT, or the S307A mutant. Cell lysates were collected and probed with antibodies indicated. (J). Gleevec sensitivity of H1703 cells transfected with SGTA siRNA. 32 hrs after SGTA siRNA transfection, cell were treated with Gleevec (1 uM) for 24 hrs and cells were counted as in 7a. Results are representative of 2 independent experiments. (K). A model for the effect of putative Akt substrates on PDGFRα stability, cancer cell growth and death.