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).