Western Blot Analysis of Cell Signaling

Western blot analysis was performed as previously described (23 (link)). Antibodies against p-Rb^Ser780, Rb, cyclin D1, CDK4 and CDK6, c-Myc, p-AKT^Ser473, AKT, p-mTOR^Ser2448, mTOR, p-p70S6K^Thr389, p70S6K, p-AMPKα1^Thr172, p-ERK1/2^{Thr202/Tyr204}, ERK1/2, p-PKM2^Tyr105, PKM2 were from Cell Signaling Technology, Incorporated (Danvers, MA); anti-p-CDK6^Tyr24 and anti-MCT4 were from Santa Cruz Biotechnology, Incorporated (Dallas, TX). Antibodies against CDKN2A/p16^INK4a, AMPKα1, GLUT-1 were from Abcam (Cambridge, UK). Antibodies against HIF-1α and HIF-2α were from BD Biosciences (Franklin Lakes, NJ) and Novus Biologicals, LLC (Centennial, CO), respectively. Anti-β-actin (clone B11V08) was from BioVision (Milpitas, CA). Horseradish peroxidase-conjugated secondary antibodies and the chemiluminescence system were from Millipore (Millipore, MA). Reagents for electrophoresis and blotting analysis were from BIO-RAD Laboratories (Hercules, CA). For GLUT-1 detection, cells were lysed in GLUT-1 lysis buffer (1 % Triton X-100, 0.1% SDS, protease inhibitors) for 1 h on ice, and precleared by centrifugation for 10 min at 4°C. Protein extracts were denatured in sample buffer for 30 min before electrophoresis (24 (link)). The chemiluminescent signal was acquired by C-DiGit R Blot Scanner and the bands were quantified by Image Studio™Software, LI-COR Biotechnology (Lincoln, NE).