Las 3000 system

Whole‐cell extracts were obtained using RIPA buffer composed of 50 mM Tris–HCl (pH 7.5), 15 mM NaCl, 1% NP‐40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate and protease inhibitors. In the Western blotting analysis, 30 μg of total protein was loaded onto a 12.5% sodium dodecyl sulfate‐polyacrylamide gel 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 following primary antibodies (Abs): anti‐IDH2 (1:500 dilution), anti‐Hif‐1α (1:200 dilution, Santa Cruz Biotechnologies, Dallas, TX, USA), anti‐PHD2 (1:500 dilution, Invitrogen, Waltham, MA, USA), anti‐GLUT1 (1:200 dilution), anti‐TIGAR (1:1,000 dilution), anti‐anti‐TKT (1:2,500 dilution), anti‐CTPS1 (1:1,000 dilution), anti‐PKM2 (1:1,000 dilution), anti‐LDHA (1:2,000 dilution), anti‐IDH1 (1:500 dilution; Abcam, Tokyo, Japan), anti‐ME1 (1:500 dilution), and anti‐G6PD (1:100 dilution). The blots were incubated with a peroxidase‐labeled secondary Ab for 1 h. After PBS washing, the signals were detected using enhanced chemiluminescence reagents with the ECL plus Western Blotting Detection System and analyzed using the LAS 3000 system (GE Healthcare, Los Angeles, CA, USA).

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).