Myosin heavy chain (MHC) gel electrophoresis

The muscles were extracted on ice for 60 min in four volumes of extracting buffer (pH 6.5), as previously described (Agbulut et al, 2003). Following centrifugation, the supernatants were diluted 1:1 (vol/vol) with glycerol and stored at −20°C. MHC isoforms (MHC‐1, MHC‐2a, MHC‐2x, MHC‐2b) were separated on 8% polyacrylamide gels, which were made in the Bio‐Rad mini‐Protean II Dual slab cell system. The gels were run for 31 h at a constant voltage of 72 V at 4°C. Following migration, the gels were silver stained. The positions of the different MHC bands were confirmed by Western blot analysis using antibodies directed against different MHC isoforms. The gels were scanned using a video acquisition system.