Anti β actin

The treated cells were collected and lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology) and incubated for 30 min on ice. Then proteins were detected using a BCA protein assay kit (Bio-Rad Laboratories, Inc.). A total of 40 µg protein was loaded onto 10% SDS-polyacrylamide gels to separate various proteins, which were subsequently transferred to PVDF membranes. The membranes were blocked with 10% skim milk for 2 h at room temperature, followed by incubation with primary antibodies for one night at 4 °C. Subsequently, the membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated IgG secondary antibodies (1:5,000; AA24142) at room temperature for just 1 h. The signals were detected using enhanced chemiluminescence reagent (GE Healthcare) and Image J software (version 146; National Institutes of Health, Bethesda) was used to analyze fold-changes of protein levels. Anti-Beclin1 (1:1,000; E2200675), anti-ATG7 (1:1,000; E90690), anti-FOXO3 (1:1,000; E0220202), anti-p62 (1:1,000; E2510713), anti-β-catenin (1:1,000; 13-8400, RRID:AB_2533039), anti-Cyclin-D1 (1:1,000; MA5-16356, RRID:AB_2537875), anti-β-actin (1:1,000; E81102-1) antibodies were obtained from EnoGene, Inc.

The cellular protein content was extracted by RIPA buffer with a protease inhibitor cocktail. Lysates were spun at 16,000 g and 4 °C for 30 min and normalized by protein concentration. The lysates were separated by SDS-PAGE and electrotransferred to nitrocellulose membranes (Invitrogen). The membranes were blocked in PBS containing 0.1% (vol/vol) Tween-20 (PBS-T) and 4% (wt/vol) nonfat dry milk (Bio-Rad) for 1 h on a shaker at room temperature. Primary antibodies were added (anti-Fumarase/FH mouse monoclonal antibody, anti-HIF-1α mouse monoclonal antibody, anti-HK2, anti-PKM2, anti-PFK2, anti-LDHA, anti-β-actin, anti-p-H2AX and anti-p-Chk2 antibodies) (EnoGene, USA) and incubated overnight on a rocker at 4 °C. Secondary antibodies were diluted 1:2,000 in blocking buffer and incubated for one hour at room temperature. Images were acquired using LabWorksTM (UVP, USA).