(A) pT308 is not inhibited by PP242 in cells overexpressing S473A Akt. HEK293 cells were transfected with wild-type Akt, S473A Akt, or not transfected (Mock) and were treated with 2.5 μM PP242 or 625 nM PIK-90 as indicated prior to insulin stimulation. Lysates were analyzed by Western blotting. Quantitation of pT308 relative to insulin treated cells overexpressing wild-type Akt (lane 2) is shown below that blot. Data are representative of two independent experiments.
(B) pT308 is not inhibited by PP242 in SIN1−/− MEFs, which lack pS473. Primary wild-type (WT) and SIN1−/− MEFs were pre-treated with 625 nM PIK-90, 10 μM BX-795, or 100 nM rapamycin for 24 h, 100 nM rapamycin for 30 min, or the indicated concentrations of PP242 prior to stimulation with insulin. Lysates were analyzed by Western blotting.