Chemidoc chemiluminescent system

Frozen muscle biopsies were disrupted with mortar and pestle and suspended in RIPA buffer (Sigma) with a proteinase inhibitor cocktail (Roche) and incubated for 30 min on ice with intermittent vortexing. Samples were centrifuged at 16,000 x g for 30 min at 4°C and the supernatant was isolated and quantified with a bicinchoninic acid assay (Pierce). Protein isolate was mixed with NuPAGE loading buffer (Invitrogen) with 5% β-mercaptoethanol and boiled at 100°C for 10 min. Samples were flash frozen in liquid nitrogen and stored at −80°C. 25 μg of total protein per lane was loaded into a 10 well 4–12% NuPAGE Bis-Tris gel (Invitrogen) with MES buffer (Invitrogen) and electrophoresed for 30 min at 200V. Protein was transferred to a nitrocellulase membrane for 1 hour at 400 mA at 4°C in transfer buffer containing 1X tris-glyceine, 10% methanol and 0.01% SDS. The blot was blocked overnight in 5% milk-TBST at 4°C. The blot was probed with MANDYS8 (1:200, Sigma D8168) and rabbit anti-GAPDH (1:5000, Cell Signaling 2118S). The blot was washed with TBST and probed with mouse or rabbit horseradish peroxidase-conjugated secondary antibodies (Sigma) for 30 min in 5% milk-TBST. Blots were visualized using Western-C ECL substrate (Biorad) on a ChemiDoc chemiluminescent system (Biorad). The full blots are shown in attached source data.