Anti hif 1α

TA muscle tissue samples were lysed in an ice-cold mixture of RIPA lysis buffer and protease inhibitor cocktail (Solarbio, Beijing, China) for 30 min. The lysate was then centrifuged (12,000 g, 10 min, 4 °C) and the supernatant was collected. The protein concentration of each supernatant was determined by the BCA protein assay (Beyotime, Shanghai, China). Each protein sample (28 μg) was then separated by 12.5 % SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). Next, 5 % non-fat milk dissolved in Tris-buffered saline containing Tween-20 (TBST) was used to block non-specific binding for 1.5 h. Next, the membranes were incubated overnight at 4 °C with a range of primary antibodies: anti-SLC7A11 (1:1000, Abcam, Cambridge, UK), anti-Gpx4 (1:1000, Abcam, Cambridge, UK), anti–HIF–1 $\mathrm{\alpha }$ (1:2000, Bioss, Beijing, China) and anti-GAPDH (1:10000, Cell Signaling Technology, Danvers, MA, USA). The following day, appropriate anti-rabbit secondary antibodies (1:1000, Proteintech, Chicago, USA) were incubated with the sections at room temperature for 1.5 h. Positive signals were then detected with an enhanced chemiluminescence detection system (Millipore, Burlington, MA, USA) and the target bands were analyzed by Image J 1.53 software (National Institute of Mental Health, USA).