Rabbit anti β actin 13e5

Total protein content was extracted by RIPA lysis buffer containing Protease and Phosphatase Inhibitor Cocktail (MedChem Express, USA) and measured using a BCA Protein Assay Reagent kit (Beyotime Biotechnology, Shanghai, China). Protein expressions were detected by Simple Western using a Size Separation Master Kit with Split buffer (12–230 kD) following the instructions of the manufacturer. The reliability of this experimental approach has been verified by previous studies [33 (link),34 (link)]. In brief, 1.5 μg protein of each sample was separated by capillary electrophoresis on Wes instrument (Protein Simple, San Jose, CA, USA) under default settings. Protein levels were probed with rabbit anti-AMPK (1:50, #2532, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-AMPK (Thr172) (1:50, #2535, Cell Signaling Technology, USA), and rabbit anti-β-actin (13E5) (1:50, #4970, Cell Signaling Technology, USA). An Anti-Rabbit Detection Module was purchased from Protein Simple. Data were analyzed by Compass software (Protein Simple, San Jose, CA, USA) version 5.0.1.