Cells or tissue were snap-frozen in liquid N2 at the end of the experiment. Cells and tissue were lysed in a modified RIPA buffer containing 50 mM Tris pH 7.5 at 4°C, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM β-glycerophosphate, 1% NP-40, 1 mM Na3VO4, 0.25% sodium deoxycholate, 0.1% SDS and protease inhibitors (Sigma). The lysate was cleared by centrifugation and the protein concentration of the cleared lysate was determined by the method of Bradford. Western blots for TORC1, TORC2, CREB and P-CREB Ser133 were performed using antibodies 6939E1, 6865, 244, 5322, respectively (Salk Institute). Antibodies for P-(Ser151) TORC1 and HSP90 were obtained from Cell Signaling and Santa Cruz, respectively.
