Fotodyne

For western-blot analysis, 30 to 50 μg of protein were used. The samples were mixed with 60 mM Tris-HCl pH 6.8, SDS, β-mercaptoethanol, and then subjected to SDS-PAGE. They were subsequently transferred to PVDF membranes for 1.5 h. Later, the membranes were blocked for 1 h, and incubated with the corresponding primary antibody: mouse anti-MHC (1:1000 MF-20; Developmental Studies, Hybridoma Bank, University of Iowa, Iowa, IA, USA), rabbit anti- β-actin (1:2000, Abcam, Cambridge, MA, USA), rabbit anti- LC3b (1:1000, Cell Signaling, Danvers, MA, USA). After the incubation time, the membranes were washed and incubated with the corresponding secondary antibody. Finally, the binding of the primary antibody / secondary antibody complex to the protein samples was detected using the chemiluminescence procedure through an image documentation system, Fotodyne (Fisher Scientific, St. Waltham, MA, USA). Densitometric analysis of the bands was performed using ImageJ software (NIH, Bethesda, MD, USA), and standardized relative to the respective load control of each sample. The MF-20 hybridoma, a monoclonal antibody developed by Fischman, D.A., was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242.

The muscle fibers were solubilized with radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate) including protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM cocktail of protease inhibitors) and scraped from the plates to prepare cell extracts. The protein concentrations were determined using a micro-BCA assay. Protein extracts from muscle fibers incubated with DCA, CA, or vehicle were obtained, mixed with loading buffer, and heated to 50 °C for 5 min. Furthermore, the samples were subjected to 12% SDS-PAGE. Electrophoretic transference was applied to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Waltham, MA, USA). The membranes were blocked with 5% skim milk–Tris buffer saline (TBS) and incubated separately with anti-OXPHOS (1:1000; Abcam, Cambridge, MA, USA) and anti-β-actin (1:1000; Abcam, Cambridge, MA, USA) antibodies. The chemiluminescent signals (Thermo Scientific, Waltham, MA, USA) were detected using secondary antibodies and peroxidase-coupled IgG (1:1000) with Fotodyne (Fisher Scientific, St. Waltham, MA, USA). The blots were then quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA) [17 (link)].

DFG, TA, and GA muscles were homogenized in Tris-ethylenediaminetetraacetic acid (EDTA) buffer (50 mM Tris, 10 mM EDTA, pH 8,3) with a cocktail of protease inhibitors (Sigma-Aldrich, St Louis, MO, USA) and 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, St Louis, MO, USA). C2C12 cells were homogenized in radioimmunoprecipitation assay (RIPA) buffer with a cocktail of protease inhibitors and 1 mM PMSF. Proteins were separated into SDS-PAGE, transferred onto polyvinylidene difluoride (PDVF) membranes (Merck, Temecula, CA, USA), and probed with rabbit anti-LC3B (1:1000, Cell Signaling, Danvers, MA, USA), rabbit anti-phospho-p38 (1:1000, Cell Signaling, Danvers, MA, USA), rabbit anti-phospho-JNK (1:1000, Cell Signaling, Danvers, MA, USA), rabbit anti-phospho-BCL-2 (1:1000, Cell Signaling, Danvers, MA, USA), rabbit anti-Beclin1 (1:1000, Cell Signaling, Danvers, MA, USA), rabbit anti-BCL-2 (1:500; Santa Cruz, Dallas, TX, USA) and mouse anti-GAPDH (1:2000; Santa Cruz, Dallas, TX, USA). All immunoreactions were visualized by enhanced chemiluminescence (Thermo Scientific, Waltham, MA, USA) which were detected through an image documentation system, Fotodyne (Fisher Scientific, St. Waltham, MA, USA).