Chemdoc mp image system

Proteins were separated using SDS-PAGE (precast Mini-PROTEAN^® TGX™ precast gels 10%, Bio-Rad) and electro-transferred to PVDF membranes (Trans-Blot^® Turbo™ Transfer System, Bio-Rad). Following transfer, the membranes were incubated in blocking buffer [Tris-buffered saline plus 0.05% Tween 20 (TTBS), supplemented with 1% BSA] for 1 h at room temperature. Primary antibodies were diluted to a concentration of 1 µg/ml in blocking buffer and applied overnight at 4°C. The blots were washed three times (10 min) in TTBS. The horseradish peroxidase-conjugated secondary antibody was diluted 1:100,000 to a concentration of 12.5 ng/ml in blocking buffer (TTBS supplemented with 2% BSA), and applied for 1 h at room temperature. The blots were then washed three times (15 min) in TTBS. Immunoreactive bands were detected and visualized by chemiluminescence using Clarity™ Western ECL substrate (Bio-Rad) and ChemDoc™ MP Image System (Bio-Rad).

Protein samples resolved on a 4%–15% TGX gel (Bio-Rad) were transferred onto PVDF membrane using standard protocol. Membranes were blocked with 2% BSA, sequentially incubated with indicated primary antibody and horseradish peroxidase-conjugated secondary antibody. Specific bands were visualized and recorded with a ChemDoc MP Image System (Bio-Rad). Primary antibodies against Ucp1, Celf1, and Celf2 were purchased from Abcam. Primary antibodies against Pparg, Pgc1α, HuR, and a-tubulin were obtained from Santa Cruz Biotechnology.

For proteins with molecular weight (MW) > 200 kDa, NuPAGE™ 3–8% tris-acetate 3–8% gels (Invitrogen, Waltham, MA, USA) and HiMark™ Pre-stained Protein Standard (Invitrogen) were used. Gels were transferred to nitrocellulose membranes via ultra-low voltage overnight transfer (12 V, ~100 mA, 20 h). For proteins with MW < 200 kDa, 10% tris-glycine gels (Invitrogen) and DualColor protein standard (Bio-rad, Hercules, CA, USA) were used instead. Primary antibodies (see Supplemental List S2 for a list of antibodies used) were diluted in 5% BSA 0.1%TBST and incubated overnight at 4 °C with gentle rocking. After primary antibody incubation, membranes were washed in 0.05% TBST three times, 10min each, followed by 1h incubation in Secondary antibody diluent. After a second series of wash, membranes were incubated in Clarity ECL substrates (Bio-rad) for 5min. Images were acquired by ChemDoc MP image system (Bio-rad).