T s6k1

Whole cell extracts were obtained using RIPA buffer composed of 50 mmol/L Tris‐HCL (pH 7.5), 15 mmol/L NaCl, 1% NP‐40, 0.5% deoxycholate, and 0.1% sodium dodecyl sulfate and containing protease inhibitors. For the western blotting analysis, 30 μg of total protein was loaded onto a 12.5% sodium dodecyl sulfate‐polyacrylamide gel, electrophoresed, and then transferred to a nitrocellulose membrane. The membrane was blocked at 4°C overnight in TBS containing 5% Phospho Blocker Blocking Reagent and 0.2% Tween‐20, and then incubated at 4°C overnight with the primary Abs for MUC1‐C (1:500 dilution), t‐AKT (1:1000 dilution, Cell Signaling, Beverly, MA, USA), p‐AKT (1:1000 dilution, Cell Signaling), t‐mTOR (1:1000 dilution, Cell Signaling), p‐mTOR (1:1000 dilution, Cell Signaling), t‐S6K1 (1:1000 dilution, Cell Signaling), p‐S6K1 (1:1000 dilution, Cell Signaling), xCT (1:500 dilution, Abcam), and MDR1 (1:250, dilution, Thermo Scientific). The blots were incubated with a peroxidase‐labeled secondary Ab for 1 h. After PBS washing, signals were detected using enhanced chemiluminescence reagents with the ECL plus Western Blotting Detection System and analyzed using the LAS 3000 system (GE Healthcare).