Myoglobin

Mouse gastrocnemius muscles were homogenized in RIPA buffer containing 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4) and protease inhibitor cocktail (Nacalai, Kyoto, Japan). Samples (1 mg) were fractionated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). Membranes were incubated with primary antibodies of the Myoglobin (1:2000 dilution, Abcam) and β-actin (1:2000 dilution, Cell signaling, Danvers, MA, USA) overnight. After incubation of secondary antibody, proteins were detected using Western Lightning Plus ECL (PerkinElmer, Waltham, MA, USA). Images were captured with ImageQuant Las 4000 and quantified by β-actin expression levels using ImageQuant TCL software (GE healthcare Life Science, Marlborough, MA, USA).

The concentrations of urinary myoglobin, titin and 3-methylhistidine (3-MH) were measured from a spot 60 mL urine sample at each timepoint (pre, immediately-post, 24-h-post and 40-h-post). Urine samples were refrigerated at 4°C immediately on receipt prior to being pipetted into three 5 mL cryotubes and stored at −80°C. The samples were then transferred to Anglia Ruskin University, Cambridge, UK for analysis. The concentrations of myoglobin (Abcam, Cambridge, United Kingdom), titin (MyBioSource, San Diego, United States) and 3-MH (Abbexa, Cambridge, United Kingdom), were quantified by an Enzyme-Linked Immunosorbent Assay (ELISA) following the manufacturer’s protocol (34–36 (link)). The inter and intra assay coefficient of variation (CV) was 6.4 and 3.4% for myoglobin, 9.9 and 9.7% for 3-MH and 8.1 and 7.8% for titin, respectively.

Wild-type C57BL/6 male mice were purchased from (Orient-Bio, Seongnam, Korea). Fetal bovine serum (FBS), horse serum (HS), and Dulbecco modified Eagle's medium (DMEM) were purchased from Thermo Scientific (Waltham, MA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DEX, and all other chemicals were from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were purchased as follows: myosin heavy chain (MHC, Developmental Studies Hybridoma Bank (DSHB), Iowa, IA, USA) Myogenin, Myoglobin, total-OxPHOS (Abcam, Cambridge, MA, USA), total-Akt, phospho-Akt, phospho-mTOR, mTOR, phospho-S6K, S6K (Cell Signaling Technology, Beverly, MA, USA), MuRF1, Atrogin-1, HSP90 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-tubulin (Zymed, South San Francisco, CA, USA).