FIG. 3.

PI3K-dependent Akt activation in 208F cells transformed by JSRV or ENTV Env proteins. (A) In vitro Akt kinase assay. Cells were incubated in serum-free DMEM overnight. The cells were then incubated for 4 h in DMEM containing 0, 25, or 50 μM LY or 50 μM LY plus 5% FBS (lane labeled “50*”). Akt activity was measured by incubating the immunoprecipitated Akt with substrate GSK-3 in the presence of 200 μM ATP, followed by immunoblotting with antibody against phospho-GSK-3α/β. Total Akt resulting from the same immunoprecipitations were determined by anti-Akt antibody. (B) Detection of Akt phosphorylation. Lysates of serum-starved cells were subjected to SDS-PAGE followed by immunoblotting with anti-phospho-Akt (Ser473) to detect Akt phosphorylation or using anti-Akt to determine the total amount of Akt present.