Calcineurin a

Protein levels were measured using western blot analysis, with β‐actin used to ensure equal loading of the samples, according to previously published details 33, and in Supporting Information Material and Methods 1. Primary antibody dilutions used were as follows; SYS, SYP, BDNF, CamK‐II, CamK‐IV, Calcineurin A (Abcam, USA), ERK1/2, p‐ERK1/2 (Cell Signaling Technology, MA, USA), and β‐actin (Santa Cruz).

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).

Cells were treated with different concentrations of As₂O₃ (2 μM or 4 μM), CsA, or NS for 72 hours. Proteins were extracted using RIPA lysis buffer, and were quantified using a bicinchoninic acid assay (BCA) protein assay kit (Thermo, Rockford, IL, USA). Equal amounts of protein (20 μg of each sample) were electrophoretically resolved on polyacrylamide gels, and then were transferred onto PVDF membranes. The membranes were blocked with a solution containing 5% nonfat dry milk for 1 hour, and were incubated with primary antibodies overnight at 4°C. After being washed 3 times with TBST (Tris buffered saline with Tween20, pH 8.0), membranes were incubated with the appropriate secondary antibody at room temperature for 1 hour, and were visualized using the enhanced chemiluminescence (ECL) detection reagents. β-actin was used as an internal control. The primary antibodies used were as follows: calcineurin A (1: 2000, Abcam, Cambridge, UK), DSCR1 (calcipressin-1) (1: 1000, Abcam, Cambridge, UK), NFAT2 (1: 500, Abcam, Cambridge, UK), RND1 (1: 500, Abcam, Cambridge, UK), CXCR7 (GPCR RDC1) (1: 250, Abcam, Cambridge, UK), and β-actin (1: 1000, Santa Cruz, Dallas, TX, USA).