Protein Expression Analysis in C2C12 Cells

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.