Quantitative Western Blot Analysis

Western blot analysis was performed as described [36 (link)]. Equal amounts of protein (15–20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membrane using Criterion XT Precast gels (Bio-Rad, USA). Membranes were blocked with 5% BSA for 2 h; then the corresponding antibodies GLUT4 (1 : 1000, #347063, Zen Bio), HK2 (1 : 1000, #200569, Zen Bio), PFKM (1 : 1000, #ab154804, Abcam), PKM (1 : 1000, #200667-1A7, Zen Bio), LDHA (1 : 500, #384822, Zen Bio), AMPKα (1 : 1000, #ab207442, Abcam), p-AMPKα (1 : 1000, #AP0116, ABclonal), mTOR (1 : 1000, #380411, Zen Bio), p-mTOR (1 : 1000, #5536, Cell signalling), 4EBP1 (1 : 500, #306002, Zen Bio), p-4EBP1 (1 : 500, #ab278686, Abcam), and Actin (1 : 2000, #200068-8F10, Zen Bio) were incubated overnight at 4°C; finally, blots were incubated with secondary antibodies at room temperature for 2 h. Bands were visualized with the gel imaging system (Syngene, UK) and analyzed with ImageJ analysis software (National Institutes of Health, https://rsb.info.nih.gov/ij/). The research met all the requirements of ARRIVE checklist and most of the requirements of CONSORT checklist if applicable (see supplemental files, named S1 ARRIVE Checklist and S2 CONSORT 2010 Checklist).