Mb001h

Proteins were extracted from iBAT using RIPA buffer (P0013B, Beyotime Biotechnology, Shanghai, China) with phosphatase and protease inhibitors (78442, Thermo Fisher Scientific, Waltham, MA, USA) and quantified in a BCA assay (23227, Thermo Fisher Scientific, Waltham, MA, USA). After extraction with 100% and 80% cold acetone, consecutively, proteins were resuspended in a loading solution (1610737, Bio-Rad, Hercules, CA, USA) with 2.5% (v/v) β-mercaptoethanol, heated at 95 °C for 3 min, separated by SDS-PAGE, and transferred onto PVDF membranes. Western blotting was conducted with antibodies against: phospho-p38 Mapk (1:1000, 4511T, Cell Signaling Technology, Danvers, MA, USA), p38 Mapk (1:1000, 8690T, Cell Signaling Technology, Danvers, MA, USA), Pgc1α (1:500, sc517380, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Ucp1 (1:500, U6382, Sigma, Burlington, MA, USA), and Gapdh (1:10000, MB001H, Bioworld, Nanjing, China). HRP-labeled secondary antibodies against rabbit (Bs13278, Bioworld, Nanjing, China) and mouse (20140714, Abgent, San Diego, CA, USA) Ig were used. Protein bands were visualized using chemiluminescent reagents (P10300, NCM Biotech, Suzhou, China) and scanned with an image analyzer (Amersham Imager 600, GE Healthcare, Chicago, IL, USA) followed by densitometric quantification.