Figure 1

Rictor depletion reduces AKT HM phosphorylation and inhibits AKT kinase activity. (A) MCF7 and PC3 cells were treated for five days with two independent siRNAs against rictor or with a control siRNA not targeting any human protein. Cells were lysed by boiling in sample buffer and proteins resolved by SDS-PAGE. Levels of Rictor expression and AKT phosphorylation and expression were analyzed by immunoblotting with anti-Rictor, anti-AKT and antibody specific for the phosphorylated form of the HM site, P-S473. (B) Cells were treated for five days with rictor siRNA, treated with insulin (Ins) or wortmannin (W) or left untreated (C). Cells were lysed and AKT was immunopurified with immobilized AKT antibodies. To measure AKT kinase activity, the immunoprecipitates were incubated with paramyosin-fused GSK-3 crosstide in the presence of ATP. The level of phosphorylation was analyzed by blotting with antibodies specific to phospho-GSK-3. Total GSK-3 level is shown as a loading control.