Fig. 6. Differential signaling to the PI3K/Akt and RhoA pathway. (A) Akt phosphorylation. 50 µg of RIPA lysates from mock-transfected CEF or cells expressing Src^527F or the chimeric constructs were resolved by 8% SDS-PAGE, transferred to PVDF membrane, blocked with 5% nonfat milk/TBS-T, and probed with an anti-phospho-Akt antibody (top panel) or an anti-Akt antibody to determine relative protein levels (bottom panel). (B) PI3K assay. 400 µg of lysates from mock-transfected CEF or cells expressing Src^527F or Y4U32^527F were immunoprecipitated with an anti-PI3K antibody and subjected to PI3K assay as described in Materials and Methods. Kinase assay products were resolved by thin layer chromatography and visualized by Phosphorimager analysis (top panel). Anti-PI3K immunoprecipitates were also resolved by 8% SDS-PAGE, transferred to PVDF membrane, blocked in 5% nonfat milk/TBS-T, and probed with an anti-PI3K p85 antibody to verify that there were equal amounts of PI3K in the lysates (bottom panel). (C) RhoA-GTP assay. CEF cells expressing the chimeric constructs or Src^527F were lysed and 500 µg of cell lysate processed for affinity absorption with GST-Crib, expressing the Crib domain of rhotekin.