Wide range molecular weight marker

SM from porcine brain, DOPC and DHPC were purchased from Avanti Polar Lipids (Alabaster, AL, USA). The soluble lipid dehydroepiandrosterone was obtained from TCI (Tokyo, Japan). The disodium salt of 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and p-xylene-bis-pyridinium bromide (DPX) were purchased from Molecular Probes (Eugene, OR, USA). Bovine serum albumin and detergent LDAO were obtained from Sigma-Aldrich (St Louis, MO, USA). DDM was obtained from Dojindo (Tokyo, Japan). Triton X-100 was purchased from Anatrace (Maumee, OH, USA). Wide-range molecular weight marker was from Bio-Rad (Hercules, CA, USA). PK was purchased from Boehringer Ingelheim (Ingelheim, Germany). Reagents for the crystallization of proteins were obtained from Hampton Research (Aliso Viejo, CA, USA) or Molecular Dimensions (Newmarket, UK). Other chemicals were from Wako (Tokyo, Japan).

For immunoblotting, 75 μg of total protein per sample were loaded into each lane for PGC-1α and GLUT4 quantification. Three controls were run simultaneously with each gel: a wide-range molecular weight marker (Bio-Rad) and total protein isolated from adult rat heart and liver. For each gel, at least one sample per group was included and grouped according to muscle type. Total protein was separated on 7.5% gels for 20 min at 80 V followed by 125 V for 1 h and then transferred to PVDF membranes for 1 h at 100 V. Membranes were blocked in 5% non-fat dry milk in PBS-T for 0.5 h then incubated with either a rabbit anti-GLUT-4 (Milipore Inc., Billerica, MA; 1:600) or a rabbit anti-PGC-1α (Abcam, Cambridge, MA; 1:1000) antibody overnight at 4°C. After washing 3 × 10 min in PBS-T, the membranes were incubated in an anti-rabbit IgG-HRP (Jackson Immunology, West Grove, PA; 1:1000) secondary antibody. Membranes were developed using Thermo Scientific Pierce ECL Western Blotting Substrate. Densitometry and quantification were completed using the ChemiDoc MP Imaging system (Bio-Rad) and Image J software (Rasband, 1997–2007 ).