2.7. Protein quantification by the Wes Simple Western System

Protein quantification was performed with the Wes Simple Western System (Proteinsimple, San Jose, CA, USA). Proteins extracted from longissimus dorsi muscle samples were mixed with fluorescent standards, Master Mix, dithiothreitol, and Simple Western Sample Buffer (Proteinsimple) and then were loaded into Wes 25-well plates. Primary antibodies against AMP-activated protein kinase (AMPK) and myocyte enhancer factor 2D (MEF2D) were purchased from Bioss, Beijing, China; antibodies against β-actin, phopho-AMPK, forkhead transcription factor 1 (FOXO1), slow skeletal myosin heavy chain (SSMHC), fast skeletal myosin heavy chain (FSMHC), Calcineurin A, Calcineurin B, peroxisome-proliferator-activated receptor coactivator (PGC-1α), and nuclear factor of activated T cells-1 (NFAT1) were purchased from Abcam, Cambridge, MA, USA. The appropriate secondary antibodies, stacking and separation gel matrix were added according to the manufacturer's instructions. Protein bands were obtained using the “gel view” function of the Protein Simple software (Proteinsimple).