Phospho rictor

Differentiated hMADS cell lysates were extracted, transferred onto nitrocellulose membranes and blotted with the following primary antibodies (all from Cell Signalling Technology Inc., Beverly, MA): phospho-Akt Ser473 (#4060), phospho-Akt Thr308 (#2965), Akt (#4691), phospho-IRS1 Tyr612 (#44816G), IRS1 (#3407), phospho-AS160 Thr642 (#4288), AS160 (#2670), phospho-p38 MAPK Thr180/Tyr182 (#9211), and p38 MAPK (#9212), phospho-Raptor Ser792 (#2083), Raptor (#2280), phospho-Rictor Thr1135 (#3606), Rictor (#2140), phospho-mTOR Ser2448 (#2971), mTOR (#2972). Immunoreactive proteins were detected by enhanced chemiluminescence reagent (SuperSignal West Dura or SuperSignal West Femto; Thermo Scientific), visualized using the ChemiDoc MP Imaging System and data analyzed using the Image Lab 4.1 version software (Bio-Rad Laboratories, Hercules, USA). α-tubulin (Sigma-Aldrich) was used as internal control.

All the antibodies were from Cell Signaling Technology (Danvers, MA, USA): mTOR (cat #2983), phospho-mTOR (Ser2481, cat #2974), phospho-mTOR (Ser2448, cat #5536), Rictor (cat #9476), phospho-Rictor (Thr1135, cat #3806), Raptor (cat #2280), AKT (cat #4691), phospho-AKT (Ser473, cat #4060), phospho-AKT (Thr308, cat #13038), phospho-AKT (Thr450, cat #9267), GSK-3β (cat #9315), phospho-GSK-3β (Ser9, cat #9336), PTEN (cat #9559), β-actin (cat #4970), 4E-BP1 (cat #9644), phospho-4E-BP1 (Thr37/46, cat #2855), S6K1/p70S6K (cat #9202), phospho-S6K1/phospho-p70S6K (Thr389, cat #9234), Myc-Tag (cat #2276), horseradish peroxidase-linked anti-rabbit IgG (cat #7074), horseradish peroxidase-linked anti-mouse IgG (cat #7076). Lipofectamine LTX, plus reagent, culture medium were from Invitrogen (Carlsbad, CA, USA). Antibiotic–antimycotic, propedium iodide (PI), rapamycin, GSK3β inhibitor (SB212763), PI3K inhibitor (LY294002 and wortmannin) and other chemicals were from Sigma–Aldrich (St Louis, MO, USA). Protease and phosphatase inhibitor cocktails were from Calbiochem (San Diego, CA, USA). Cycle Test Plus kit was from BD Bioscience (East Rutherford, NJ, USA). Super Signal West Pico imaging system was from Thermo Scientific (Rockford, IL, USA).

Resveratrol, anti-β-actin antibody, and the c-Myb inhibitor, celastrol, were purchased from Sigma-Aldrich. For Western blotting, monoclonal antibodies against phospho-STAT5, STAT5, phospho-Akt, Akt, phospho-Erk, Erk, phospho-PDK1, PDK1, phospho-rictor, rictor, and c-Myb were purchased from Cell Signaling Technology (Danvers, MA, USA).