Cells were harvested and disrupted in a Sample Buffer Solution with 2-ME (Nacalai Tesque Inc.). Samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis using a 5–20% Extra PAGE One Precast Gel (Nacalai Tesque Inc.) and electrotransferred to a polyvinylidene fluoride membrane with the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Hercules, CA; 2.5 A, 25 V, 7 min). The membrane was blocked for 1 h in 5% (w/v) skimmed milk in Tris-buffered saline containing 0.05% (v/v) Tween 20, then incubated with primary antibodies in Hikari Solution A (Nacalai Tesque Inc.), followed by incubation with secondary antibodies and detection using Chemi-Lumi One Ultra (Nacalai Tesque Inc.).
The following primary antibodies were used for western blotting: rat anti-MyoD (5F11, Millipore 1 : 500), anti-Mettl3 (15073-1-AP, Proteintech, 1 : 1000), anti-Hsp90 (H-114, Santa Cruz Biotechnology, 1 : 1000) and mouse anti-HuR (3A2, Santa Cruz Biotechnology, 1 : 200). Secondary antibodies were horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG antibodies (GE Healthcare, 1 : 5000).
The following primary antibodies were used for western blotting: rat anti-MyoD (5F11, Millipore 1 : 500), anti-Mettl3 (15073-1-AP, Proteintech, 1 : 1000), anti-Hsp90 (H-114, Santa Cruz Biotechnology, 1 : 1000) and mouse anti-HuR (3A2, Santa Cruz Biotechnology, 1 : 200). Secondary antibodies were horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG antibodies (GE Healthcare, 1 : 5000).