Protein Isolation and Western Blot Analysis

Total protein was isolated from cells or tissues using RIPA buffer supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The protein concentration was determined with the BCA protein assay (Thermo Fisher Scientific, USA). Protein separation and western blot analysis were conducted as described earlier [5 (link)]. GADD45A antibody (sc-6850, 1: 1,000) was from Santa Cruz Biotechnology (Santa Cruz). Perilipin-1 (ab61682, 1:2000) and UCP1 (ab10983, 1:1000) were from Abcam. FABP4 (E71703-98, 1:2000) and GAPDH (EM1101, 1:5000) were from HuaBio. Cocktail (45-8099, 1:2000) was from Thermo Fisher Scientific. Stat1 (D4Y6Z, 1: 1000) and Phospho-Stat1 (Tyr701) (58D6, 1:1000) were obtained from Cell Signaling Technology. The horseradish peroxidase (HRP)-conjugated secondary antibody (anti-rabbit IgG, 111-035-003 or anti-mouse IgG; 115-035-003, Jackson ImmunoResearch) was diluted 1:10,000. Immunodetection was performed using enhanced chemiluminescence western blotting substrate (Google Biotechnology, Wuhan, Hubei, China) and detected by ChemiScope3500 Mini System.