Anti wnt5a

According to previous established procedures, cells and muscle samples were harvested, washed with 1× phosphate‐buffered saline (PBS), and lysed in NP40 lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 0.1% NP‐40, 5 mM EDTA, 10% glycerol) with protease inhibitors cocktail (Sigma‐Aldrich, St. Louis, MO, USA).18 Proteins were separated in SDS‐PAGE, transferred, and immunoblotted with various antibodies. The antibodies used were anti‐Wnt5a (1:1500; Invitrogen, Carlsbad, CA, USA), anti‐MyoD (1:1500; Invitrogen), anti‐MyoG (1:1500; Invitrogen), anti‐MEF2C (1:1500; Invitrogen), anti‐Myf5 (1:1500; Invitrogen), and anti‐β‐actin (1:3000; Santa Cruz Biotechnology, Dallas, TX, USA).

Chopped tissues and cells were placed in radioimmunoprecipitation assay lysis buffer containing 1% phenylmethylsulfonyl fluoride (Beyotime, Jiangsu, China). Supernatant was collected after 12000 r/min for 15 min. Protein lysates were separated by gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were exposed to anti-Wnt5a (Invitrogen) or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies at 4°C overnight. The membrane was incubated in the anti-rabbit IgG secondary antibody (1:5000; Proteintech, Rosemont, IL, United States) for 2 h at room temperature. A ProtoBlot II AP System (Promega, Madison, WI, United States) was used to test the signals. The protein expression was compared with the expression of GAPDH. Densitometry quantified the protein relative expression with ImageJ software (National Institutes of Health, Bethesda, MD, United States).