FIGURE 4.

The PI3K-mediated decrease in SARA is independent of Akt activity and may involve direct SARA interaction with p85α. A, HKC were transfected with either empty vector (EV) or AktK179M for 3 h prior to treatment with either DMSO vehicle (V), or 20 μm LY294002 (LY) for an additional 16 h and then lysed for Western blot. β-Actin is included as a control for loading. B, HKC were transfected with empty vector or AktK179M along with β-galactosidase and the αSMA promoter and treated as in A followed by lysis and luciferase β-galactosidase assays to control for transfection efficiency. LY294002 induced αSMA promoter (*, p = 0.0003), and AktK179M did not (p = 0.3168). C, HKC stably expressing either control (sh-cont) or (sh-p85α) were serum-deprived and treated for 16 h with vehicle (V, DMSO) or 20 μm LY294002. β-Actin is shown to control for even loading. D, control (black bars) or sh-p85α (white bars) expressing cells were assayed for mRNA expression of either αSMA or p85α via quantitative RT-PCR. The sh-p85α cells had significantly increased αSMA mRNA (*, p = 0.02) and decreased p85α mRNA (*, p = 0.008). E, serum-deprived HKC were treated with either vehicle (V) or 20 μm LY294002 (LY) for 16 h and lysed for immunoprecipitation and immunoblotting (IB). Negative control (nc) is a lysate-free IP lane from the same experiment run on a different region of the same gels.