Fotodyne foto analyst luminary workstation systems

For the protein extracts, myotubes or TA muscles were homogenized in a radioimmunoprecipitation assay (RIPA) buffer with 1 mM of a cocktail of protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA) and 1 mM of phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA). Proteins were subjected to SDS-PAGE, transferred onto polyvinylidene difluoride membranes (Thermo Fisher Scientific, Waltham, MA, USA), and probed with anti-OXPHOS (1:1000; Abcam, Cambridge, MA, USA), anti-PGC-1α (1:1000, Cell Signaling, Danvers, MA, USA), anti-PINK-1 (1:1000, Cell Signaling, Danvers, MA, USA), anti-LC3B (1:1000, Cell Signaling, Danvers, MA, USA), anti-TOM20 (1:1000, Cell Signaling, Danvers, MA, USA), anti-GAPDH (1:2000; Santa Cruz, Dallas, TX, USA) and anti-β-actin (1:1000; Abcam, Cambridge, MA, USA) antibodies. All immunoreactions were visualized by enhanced chemiluminescence (Thermo Scientific, Waltham, MA, USA). Images were acquired using the Fotodyne FOTO/Analyst Luminary Workstation Systems (Fisher Scientific, St. Waltham, MA, USA). Densitometry analysis was determined by scanning immunoreactive bands. Intensity values were obtained for further normalization against the control group using ImageJ software (National Institutes of Health [NIH], Bethesda, MD, USA).

The ubiquitinated MHC levels were determined. Briefly, total protein extracts from TA muscles were made with the non-denaturant lysis buffer and were immunoprecipitated with anti-MHC (1:100 MF-20; Developmental Studies, Hybridoma Bank, University of Iowa, Iowa, IA, USA). The antibody/antigen complex was separated from the sample using agarose beads coupled to protein G to isolate MHC. The immunoprecipitates were separated using SDS-PAGE and transferred to a PVDF membrane, which was subsequently incubated with antibodies against ubiquitin (1:5000, Santa Cruz, Dallas, TX, USA), MHC (1:1000 MF-20; Developmental Studies, Hybridoma Bank, University of Iowa, Iowa, IA, USA), and β-actin (1:2000, Abcam, Cambridge, MA, USA). Immunoreaction was visualized using chemiluminescence reagents (Thermo Scientific, Waltham, MA, USA), in Fotodyne FOTO/Analyst Luminary Workstation Systems (Fisher Scientific, St. Waltham, MA, USA). Densitometric analysis was determined using ImageJ software (NIH, Bethesda, MD, USA).