Protein Expression Analysis in Fibroblasts

Western blot was performed following previously published method [29 (link)] to analyze the changes of specified proteins. Briefly, primary skeletal muscle fibroblasts were lyzed with RIPA buffer (Beyotime Bio, Shanghai, China) supplemented with proteinease inhibitor and phosphatase cocktail (Sigma, St. Louis, MO) and total proteins (40 μg) were resolved on 8% SDS-PAGE gels and transferred onto PVDF membranes. The membranes were blocked in 5% nonfat milk in TBST (50 mM Tris, pH 7.5; 150 mM NaCl; 0.1% Tween 20) for 45 min, incubated with primary antibodies at 4 °C overnight, washed and incubated with proper horseradish peroxidase conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) at room temperature for 60 min before visualized with enhanced chemiluminescence (ECL) reagents (Pierce, Rockford, IL). The primary antibodies used were Col I antibody, a-SMA antibody, CTGF antibody, TGF-β1 antibody, Fibronectin antibody, p-AKT antibody, AKT antibody, p-ERK antibody, PKC antibody, p-PKC antibody and β-Actin Antibody (sc-47,778) were purchased from Santa Cruz Biotech (Shanghai, China).