Myomaker

Cultured C2C12 cells were homogenized in ice-cold radioimmunoprecipitation assay (RIPA) buffer (Wako) with protease and phosphatase inhibitors (Nacalai Tesque Inc.) and processed for western blotting analyses as previously described [25 (link),26 (link)]. Primary antibodies used were against phosphorylated MyoD (1:1000; Santa Cruz Biotechnology Inc.), myogenin (1:1000; DSHB), myomaker (1:1000, Abcam, Cambridge, UK), myomerger (1:1000; R&D Systems), IL-4Rα (1:1000; Santa Cruz Biotechnology Inc.), MyHC (1:200; DSHB), and GAPDH (1:2000; Cell Signaling Technology Inc., Danvers, MA, USA). Luminescence signals from ECL reagent (Bio-Rad Laboratories Inc., Hercules, CA, USA) were detected using LAS-4000 (Fujifilm Corp., Tokyo, Japan). Quantitative densitometric analyses were performed using Fiji software.

Muscle tissues and collected myoblasts were homogenized and lysed in RIPA buffer (Thermo Fisher Scientific) containing protease inhibitor cocktails (P2714, Sigma-Aldrich). The lysates were centrifuged and 20–30 μg protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore). Immunoblot analysis was conducted using antibodies against Nox4 (Ewha Women's University), α-tubulin (Abcam), MyoD (Santa Cruz Biotechnology), myosin heavy chain 3 (MHC, Santa Cruz Biotechnology), myogenin (Santa Cruz Biotechnology), myomaker (Abcam, ab188300), and GAPDH (Santa Cruz Biotechnology). HRP-conjugated goat antibody (Santa Cruz Biotechnology) was used as secondary antibody. Immunocomplexes were visualized using Western Blotting Luminol Reagent (Bio-Rad).