Ab155117

Cells were lysed using RIPA Buffer (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail set I (EMD Millipore, Burlington, MA, USA). Protein samples were separated on 4–20% SDS-PAGE (Thermo Fisher Scientific) and transferred for 90 min at 40 V onto PVDF membranes (EMD Millipore). Membranes were blocked using Odyssey blocking buffer (Licor). Membranes were incubated with primary antibodies at 4 °C overnight then washed with PBS-Tween and incubated with secondary antibodies for 1 h at room temperature. Western blots were imaged and quantitated using the Odyssey infrared imaging system. Western blot analysis was carried out using the following antibodies: UCP1 (Abcam ab155117, 1:500), FABP4 (Abcam ab66682, 1:1000), and β-actin (Santa Cruz sc-47778, 1:1000).

Cell and tissue lysates were prepared using RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, and protease) and phosphatase inhibitor cocktail (Roche Diagnostics Co., CA, USA). Protein concentrations were measured with a BCA assay kit (Pierce Diagnostics Co., CA, USA). Protein was separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore, MA, USA). Membranes were blocked in 5% skim milk in TBST (0.02 M Tris base, 0.14 M NaCl, and 0.1% Tween 20, pH 7.4) followed by incubation with primary antibodies overnight at 4°C and then incubation with secondary antibodies conjugated with HRP. Primary antibodies used in the current study are EN1 (ab108598, R&D Systems, MN, USA), PPARγ2 (#2443, Cell Signaling Technology, MA, USA), AP2 (A0232, ABclonal Biotech Co., MA, USA), UCP1 (ab155117, Abcam Co., MA, USA), PGC1α (ab54481, Abcam Co., MA, USA), OXPHOS (ab110413, Abcam Co., MA, USA), and GAPDH (#2118, Cell Signaling Technology, MA, USA). Signals were detected with Super Signal West Pico Chemiluminescent Substrate (Pierce, IL, USA).