FIG. 5.

Differential sensitivity of mTORC1 and mTORC2 complex formation to rapamycin and 1-BtOH. (A) 786-O cells were plated at 5 × 10⁵/60-mm plate, incubated for 24 h, and then shifted to medium containing 0.5% serum. 1-BtOH (0.25%) and the indicated concentrations of rapamycin (Rap) were added as indicated. After 6 h, lysates were prepared and subjected to immunoprecipitation with anti-mTOR antibody overnight, and then the mTOR immunoprecipitate (IP:mTOR) was subjected, along with the lysates, to Western blot analysis for Rictor and Raptor. The lysates were also analyzed for levels of Akt phosphorylated at Ser473 (P-Akt S⁴⁷³), Akt, S6 kinase phosphorylated at Thr389 (P-S6K T³⁸⁹), and S6 kinase (S6K). (B) 786-O cells were prepared as in panel A and treated with 0.5% 1-BtOH and 200 nM rapamycin as indicated. PA (100 μM) was added along with 1-BtOH where indicated. After 6 h, lysates were prepared and subjected to immunoprecipitation with anti-mTOR antibody overnight and then the mTOR immunoprecipitate was subjected, along with the lysates, to Western blot analysis for Rictor. The data shown are representative of three independent experiments.