Western Blot Analysis of Muscle Proteins

Approximately 4 µg of protein, as determined by a BCA assay, was loaded onto NuPAGE Novex 4–12% BIS/Tris gels (ThermoFisher Scientific) and fractionated at 200 volts until the bromophenol blue marker was at the bottom of the gel. Gel contents were then transferred onto Pall FluoroTrans membranes (Fisher Biotec Wembley, Australia) and probed with rabbit anti-GAA antibody (Abcam, cat. no. 137068, Melbourne, Australia)^{26 (link)} at 1:1,000 dilution, anti-desmin antibody (ThermoFisher Scientific, cat. no. PA5-16705)^{62 (link)} at 1:5,000 dilution, mouse monoclonal anti-β-tubulin antibody (DSHB, cat. no. E7, Iowa City, Iowa)^{63 (link)} at 1:2,000–6,000 dilution and mouse monoclonal anti-β-actin antibody (Sigma-Aldrich, cat. no. A5316)^{64 (link)} at 1:100,000 dilution overnight at 4 °C. Polyclonal goat anti-rabbit or anti-mouse immunoglobulins/HRP (Dako, cat. no P0448 and D0447 respectively, North Sydney, Australia) at a dilution of 1:10,000 and Luminata Crescendo Western HRP substrate (Merk Millipore, Bayswater, Australia) were used for immunodetection and a serial scan of 30 s was performed using Fusion FX system (Vilber Lourmat, Marne-la-Vallée, France). The entire image was processed and densitometric analysis was performed using ImageJ (NIH; https://imagej.nih.gov/ij/download.html)^{65 (link)}.