Il 4rα

Tissues and cells were homogenized in Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Homogenates (20 μg of total protein) were separated by SDS‐PAGE and transferred to nitrocellulose membranes. Blots were probed with primary antibodies against Sirt6, GATA‐1, Ac‐K, Ac‐H3K27 (Cell Signaling, Beverly, MA, USA), PGC1α, PRDM16, CCR3, IL‐4Rα (Abcam), α‐tubulin, UCP1 (Sigma‐Aldrich), myc, HA tag (Thermo Fisher Scientific, Waltham, MA, USA), Ac‐H3K27 (Active Motif, Carlsbad, CA, USA), Ym1 (Stemcell Technologies, Cologne, Germany), and arginase 1 (Arg1, Santa Cruz Biotechnology, Dallas, TX, USA). Immunoreactive bands were detected with a Las‐4000 imager (GE Healthcare Life Science, Pittsburgh, PA, USA).

Cultured cells were washed with PBS, fixed, permeabilized with 0.3% Triton X-100 (Sigma), blocked with 1% bovine serum albumin (BSA, Sigma), and incubated overnight at 4 °C with the following primary antibodies: Pax7 (Abcam), Myod1 (Abcam), Myf5 (Abcam), Desmin (Abcam), Dystrophin (Abcam), Myogenin (Abcam), MyHC (DSHB), IL4Rα (Abcam), and PDGFRα (CST), this was followed by incubation with secondary antibodies for one hour at room temperature. The fusion index was determined as the ratio between the number of nuclei (at least two) incorporated into myotubes determined by immunodetection of MyHC and the total number. Tissue samples were sliced into serial 8-μm frozen sections. Tissue sections were fixed with cold acetone, permeabilized, and blocked as previously described, and then incubated overnight with primary antibodies against Dystrophin (Abcam), GFP (Abcam), CD206(Abcam), and INOS(Abcam) at 4 °C, followed by incubation with secondary antibodies for one hour at room temperature. Fluoroshield™ with DAPI (Sigma) was used to stain the nuclei. Immunoreactivity was visualized and imaged using a NIKANG fluorescence microscope (Olympus, Tokyo, Japan).