Western Blot Analysis
Cells were lysed in RIPA buffer containing 150mM sodium chloride, 1.0% TritonX-100, 0.5% sodium deoxycholate, 0.1% SDS, 50mM Tris with freshly added protease and phosphatase inhibitor cocktail (Roche Diagnostics Corp, Pleasanton, CA, USA). Equal amount of protein samples were distributed in 10% SDS-polyacryl-amide gels. After electrophoresis, proteins were transferred to a PVDF membrane, incubated with blocking buffer (5% fat-free milk) for 1 h at room temperature, and blotted with the following antibodies overnight: anti-PRDM16 (R&D), anti-UCP1 (Abcam), anti-PPARγ (CST) and anti-β-actin (SigmaChemical). The membrane was incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Signals were visualized using Mini ChemiTM 580 (Sage Creation Science Co, Beijing, China) with Super Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA).