Acadm

Cell and tissue extracts were prepared in lysis buffer (50 mmol/L Tris-HCl (pH = 8), 150 mmol/L NaCl, 0.2% SDS, 1% NP-40, 0.5% Deoxycholat, 1 mmol/L PMSF and protease- and phosphatase inhibitors (Roche Diagnostics, Manheim, Germany). Protein samples (10 μg per lane) were separated on 12.5% PAGE–SDS gel, transferred (Biometra, Göttingen, Germany) on nitrocellulose membranes (Whatman, Dassl, Germany), exposed to primary antibodies overnight at 4°C (ACADS, ACADM, ACADL, ACADVL, Sigma Aldrich, Steinheim, Germany; GAPDH, Ambion, Invitrogen, Karlsruhe, Germany) followed by exposure to fluorochrome-conjugated secondary antibodies (Li-COR Bioscience, Bad Homburg, Germany). For quantification of ACADS, ACADM, ACADL, ACADVL and GAPDH protein levels the Odyssey infrared imaging system (Li-COR Bioscience, Bad Homburg, Germany) was used.

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.