Immobilon p polyvinylidene difluoride membranes

Tissue extracts for protein analysis were prepared by homogenization in a buffer containing 50 mM Tris HCl (pH 7.4), 150 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 2 mM sodium orthovanadate, 10 mM b-glycerophosphate, 5 mM sodium fluoride and a protease inhibitor cocktail (cOmplete-Mini, Roche, Sant Cugat del Valle `s, Spain). Protein concentration in each sample was determined by the BCA method (Thermo Fisher Scientific). Total protein (30 mg/lane) was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% acrylamide/ bisacrylamide gels using a Mini Trans-Blot kit (Bio-Rad, Hercules, CA, USA) and transferred to Immobilon-P polyvinylidene difluoride membranes (GE Healthcare, Little Chalfont, UK). Membranes were then incubated first with primary antibodies specific for UCP1 (ab10983; Abcam) or b-actin (A5441, Sigma-Aldrich) followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (ab6721, Abcam) or anti-mouse IgG (1721011, Bio-Rad) secondary antibodies. Signals were detected using a chemiluminescence horseradish peroxidase substrate (EMD Millipore) and expressed relative to b-actin signal and total tissue abundance. Digitized images were quantified using the Multi Gauge V3.0 software suite (Fujifilm, Tokyo, Japan).