Anti actb

Total protein was extracted by total protein extraction kit (KeyGEN BioTECH, Jiangsu, China), the concentration was assessed and the samples were boiled for 5 min. Then, proteins were separated by electrophoresis and transferred to the PVDF membrane, and blocked with a blocking solution (Sigma, USA) at room temperature for two hours. Next, samples were incubated with anti-HIF-1α (1:500, Sangon Biotech, Shanghai, China) and anti-ACTB (1:500, Sangon Biotech, Shanghai, China) antibody overnight at 4 °C, and then with anti-rabbit IgG, HRP-linked antibody (1:1000, CST, USA) at room temperature for 1 hour. Finally, samples were analyzed using a gel imager.

The total protein was extracted using RIPA Lysis Buffer (Meilunbio, Dalian, China). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was chosen for the total protein separation, and the proteins were then transferred to nitrocellulose membranes (the membranes were cut horizontally). The membranes were incubated with primary antibodies, including anti-p62/SQSTM1 (Abcam (Cambridge, UK), Cat# ab207305), anti-LC3B (Abcam, Cat# ab192890), anti-ACTB (Sangon Biotech (Shanghai, China), No. D110001), anti-PARP (Proteintech (Rosemont, IL, USA), Cat No. 13371-1-AP), anti-cleaved caspase3 (Cell Signaling Technology (Danvers, MA, USA), 5A1E), anti-SIRT1 (Cell Signaling Technology (Danvers, MA, USA), C14H4), anti-Acetylated lysine (Cell Signaling Technology (Danvers, MA, USA), #9441), and anti-Beclin1 (Proteintech, Cat No. 11306-1-AP). Immunoprecipitation was carried out either by incubating Anti-Flag beads (MCE (Addison, IL, USA), Cat. No. HY-K0207) at 4  °C with lysate overnight or by incubating an appropriate antibody with cell lysate for 4–6 h, followed by incubating Protein A/G immunoprecipitation beads overnight. Immunoprecipitates were washed three times with cold lysis buffer and eluted with SDS loading buffer by boiling for 10 min. The original western blot figures can be found in Figure S1.