FIG. 1.

Phosphorylation of Akt at Ser473 in 786-O cells is dependent on mTORC2. 786-O cells were plated at 2 × 10⁵/60-mm plate. Twenty-four hours later, the cells were transfected with siRNA for Raptor, Rictor, or mTOR or a scrambled siRNA as described in Materials and Methods. Twenty-four hours later, the cells were treated with fresh medium containing 10% serum for an additional 48 h. The cells were then harvested and analyzed for levels of mTOR, Raptor, Rictor, Akt phosphorylated at Ser473 (P-Akt S⁴⁷³), Akt, S6 kinase phosphorylated at T398 (P-S6K T³⁸⁹), and S6 kinase (S6K) by Western blot analysis. All of the data shown are representative of at least three independent experiments.