Myogenin

Twenty-four hours after transfection with siRNAs, the cells were lysed with RIPA buffer (Nakarai Tesque). Samples were boiled for 10 min in NuPAGE sample buffer (Invitrogen) and were separated by SDS–polyacrylamide gel electrophoresis. The proteins were subsequently transferred onto an Immobilon-P PVDF transfer membrane (Millipore, Bedford, MA). The membranes were blocked with 3% bovine albumin (Nakarai Tesque) prepared in phosphate-buffered saline with Tween 20 (PBS-T) and then incubated with one of the primary antibodies: β-actin (1:10,000, Sigma-Aldrich, St. Louis, MO), PAX3 (1:500, R&D Systems, Minneapolis, MN), Myogenin (1:500, Novus Biologicals, Centennial, CO), and B7-H3 (1:1000, Cell Signaling Technology, Danvers, MA). The membranes were then washed with PBS-T and incubated with anti-mouse or anti-rabbit secondary antibody (1:10,000, Santa Cruz Biotechnology, Santa Cruz, CA). Antibody binding was detected using an ECL prime detection system (GE Healthcare, Buckinghamshire, UK).

About 100 μg of protein derived from crushed muscle extracts and uninjured muscle were run out on a separate acrylamide gel, transferred to PVDF membrane (BioRad, Mississauga, Ontario), blocked with 5% skim milk for 1 h at RT, and then incubated overnight at 4°C with primary Active-HGF antibody (Abcam). A similar western blot protocol was completed for the analysis of c-met (Sigma Aldrich) and myogenin (Novus Biologicals) protein content, using 30 μg of protein from lysates of uninjured and 5 days post-injury muscles, respectively. The appropriate horseradish peroxidase-conjugated secondary antibodies were incubated for 1 h at RT, and the blot was visualized using SuperSignal Chemiluminescent reagent (Thermo Scientific). Images were acquired using a Gel Logic 6000 Pro Imager (Carestream, Rochester, NY) and the area density of each band was analyzed using Adobe Photoshop.