Fabp4

Cells were lysed with RIPA buffer (Biocolors Biology, Shanghai, China). The protein concentration was assayed using a Pierce BCA Protein Assay Kit (Thermo Scientific, USA). The boiled samples were separated by SDS-PAGE and electrotransferred to PVDF membranes (Millipore, USA). The immunoblots were blocked with 10% nonfat milk and incubated with antiantibodies for UCP1, β-ACTIN (Santa Cruz, USA), PGC-1α, PPARγ, FABP4, and FAS (Cell Signaling Technology, USA) overnight at 4°C. After that, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, USA), and target protein bands were detected using an enhanced chemiluminescence system (Millipore, USA). Anti-β-ACTIN antibodies were employed as internal control total cellular proteins.

Cells were washed with PBS and lysed in 10 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, protease inhibitor, 1 mM sodium orthovanadate, and phosphatase inhibitor cocktail for 30 min at 4 ˚C. Isolated cell extracts were separated on SDS-PAGE and transferred to nitrocellulose membrane (Whatman, Clifton, NJ, USA). After blocking nonspecific binding sites with 5% nonfat milk at RT for 1 hr, membranes were incubated with primary antibodies overnight at 4 ˚C. Antibodies to C/EBPα, C/EBPβ, PPARγ, FABP4, Perilipin, p-AKT(Ser473), and AKT were obtained from Cell Signaling Technologies (Berkeley, CA, USA) and antibodies to Hsc70 were obtained from Rockland (Gilbertsville, PA, USA).The immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies. Protein detection was performed by using enhanced chemiluminescence (Pierce, Rockford, IL, USA). Nuclear fraction was extracted using a nuclear extract kit (Pierce) according to the manufacturer’s recommendation.