Goat anti rabbit and goat anti mouse secondary antibodies

Cold RIPA buffer (50 mM Tris pH 8.0, 1% non-idet P40, 0.5% deoxycholate, 0.1% SDS, 150 mM NaCl) supplemented with 10 μL/mL phosphatase inhibitor cocktail 3 and cocktail 2 (Sigma, St. Louis, MO) was used to harvest the total protein extract. Protein concentration was determined by using Pierce^TM BCA protein assay kit (# 23227, Thermo Scientific) based on absorbance assay. 20 μg of total protein extract from indicated experimental condition was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA) and transferred to polyvinylidene difluoride membranes (Bio-Rad). Non-specific binding was blocked in non-fat milk (5%) before the membranes were incubated with specific primary antibodies at 4°C overnight followed by appropriate secondary antibodies for 1 h at room temperature. Primary antibodies used included PGC-1α (1:1000), PPARα (1:1000), ERRα (1:1000), ACADM (1:1000), and GAPDH (1:3000), all from Sigma. Goat-anti-rabbit and goat-anti-mouse secondary antibodies were purchased from Bio-Rad and donkey-anti-goat secondary antibody was purchased from R&D (Minneapolis, MN). Signals were detected by enhanced chemiluminescence (GE Healthcare, Buckinghamshire, UK) and exposure to on X-ray film. Films from at least three individual experiments were scanned and densitometric analysis was performed with ImageJ software.

Compounds C1-C6 were synthesized by the laboratory at the Ocean University of China, Qingdao, China. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). All compounds were dissolved in DMSO at stock concentrations of 80 mM and stored at 4°C. Fetal bovine serum (FBS) was purchased from Aleken Biologicals (Nash, TX, USA). Dulbecco’s modified Eagle’s medium/F12 medium and penicillin/streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Rabbit polyclonal antibody against human poly(ADP-ribose) polymerase (PARP; H-250), mouse monoclonal antibodies against Ub (P4D1), Bax (B-9), goat polyclonal antibody against β-actin (C-11) and donkey anti-goat secondary antibody were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Goat anti-rabbit and goat anti-mouse secondary antibodies were from Bio-Rad (Hercules, CA, USA).