B cell lymphoma 2 bcl 2

Cells were lysed using RIPA lysis buffer (Biosharp, Beijing, China) with a protease inhibitor cocktail (Cell Signaling Technology, Boston, MA, USA). Total soluble protein was electrophoresed from 8% to 12% on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). After blocking with 5% albumin from bovine serum (BSA) or skimmed milk for 1 h at room temperature, the blots were probed with primary antibodies to GLUT1 (dilution 1:1000, ABclonal, Wuhan, China), B-cell lymphoma-2 (Bcl-2, dilution 1:5000, Proteintech, Rosemont, IL, USA), BCL2-Associated X (Bax, dilution 1:1000, Proteintech, Rosemont, IL, USA), p-Akt (Ser473), total Akt (dilution 1:1000, ABclonal, Wuhan, China), p-mTOR (Ser2448) or total mTOR (dilution 1:1000, Santacruz Biotechnology, Dallas, TX, USA) and kept overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibodies (Biofly, Chengdu, China) for 1 h at room temperature. After washing 3 times with 1% TBST, the bands were visualized using an efficient chemiluminescence (ECL) kit (Millipore, Burlington, MA, USA). β-actin (Sigma, Saint Louis, MO, USA) was used as the loading control.