Immunoblotting

Immunoblotting was performed as previously described.¹¹ Briefly, 10-μm frozen muscle sections were homogenized in 4% SDS lysis buffer and assessed for total protein concentration using the Pierce BCA protein assay kit (ThermoFisher Scientific). Proteins were separated by SDS-PAGE using precast minigels (Invitrogen) or criterion gels (Biorad), then transferred to polyvinylidene fluoride membranes (PVDF, Millipore). These were blocked with 5% skim milk in 1× PBST, probed with probed antibodies (Supplemental Material and Methods) overnight and secondary antibodies at room temperature for 2 hr, then developed with ECL reagents (Amersham Biosciences). Membranes were washed, developed, and imaged by Image Quant (GE Healthcare). Densitometry was performed using ImageJ image processing software (NIH) and quantified using the area under the curve. This was normalized to myosin and actin expression, obtained from PVDF membranes stained with Coomassie brilliant blue (1610436, Sigma-Aldrich).