Gb11767c

We used the BCA assay kit to determine the protein concentrations and separated proteins of total extracts on 10% SDS gels (W003-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Separated proteins were electrophoretically transferred onto polyvinylidene difluoride membrane (PVDF) (WGPVDF45, Servicebio, Wuhan, China) and blocked with 5% non-fat milk in phosphate-buffered saline (PBS)-Tween for 2 h. The primary antibodies were polyclonal rabbit anti-Bcl-2 (1:1000, GB124830, Servicebio, China), anti-BAX (1:1000, GB12690, Servicebio, China), anti-cleaved-caspase 3 (1:1000, GB11767C, Servicebio, China) and β-actin (1:2000, GB11001, Servicebio, China). They were incubated with the membranes at 4 °C overnight. The membranes were incubated with a secondary antibody conjugated to horseradish peroxidase (Beyotime, Shanghai, China) after being washed with TBST three times for 10 min each time. Bands were visualized using an ECL system (G2014-50ML, Servicebio, China). The ChemiDoc-It2 610 imaging system (UVP, LLC, Upland, CA, USA) was used to conduct the visualization, and Image-Pro Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD, USA) was used for quantification. All experiments were independently replicated three times.

For protein extraction, the samples were homogenized in tissue protein extraction reagent. Protein concentration of supernatants was determined by the BCA protein assay kit. The 20 mg of protein per sample was separated via 10% SDS‐PAGE and transferred onto PVDF membranes. Afterward, the proteins were then incubated overnight at 4°C with different primary anti‐bodies: anti‐CD31 (Bioss, bs‐0915R, 1:1000), anti‐CK18 (Servicebio, GB11232, 1:1000), anti‐Vimentin (Servicebio, GB1192, 1:1000) or anti‐Caspase 3 (Servicebio, GB11767C, 1:1000). Membranes were then washed three times with TBST and incubated with the HRP‐conjugated anti secondary antibody. The ImageJ software was used for densitometric analyses of protein bands.

Total protein was purified from satellite cells using radioimmunoprecipitation assay (RPMI) buffer (Beyotime, Shanghai, China), and electrophoresed on an 8−12% sodium dodecyl sulfate polyacrylamide gel, and transferred to polyvinylidene fluoride membranes (Millipore Sigma, Burlington, MA, USA). Next, the membranes were blocked with 5% (w/v) bovine serum albumin and incubated with primary antibodies against Caspase-3 (GB11767c; Servicebio, Wuhan, China), Caspase-7 (27155-1-ap; Proteintech, Chicago, IL, USA), Caspase-9 (GB11053-1; Servicebio, Wuhan, Hubei, China), BCL2 (BF9103; Affinity Biosciences, OH, USA), and β-actin (8227; Abcam, Cambridge, MA, USA), and visualized using an ECL Western blot detection kit (Thermo Scientific, Rockford, IL, USA) and ImageQuant LAS 4000 (GE Healthcare, Piscataway, NJ, USA).