Anti ucp1

The adipose tissues were triturated, and RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitors was added. The samples were then incubated on ice for 30 min. Total protein concentrations were determined using a BCA protein analysis kit (Beyotime, Shanghai, China). Next, 25 µg of each protein was loaded, and the samples were separated by 12% SDS-PAGE (Bio-Rad). The resulting protein bands were then transferred to PVDF membranes for Western blotting. The PVDF membranes were blocked for 1 h at room temperature in 5% skimmed milk. Next, the membranes were incubated with primary antibodies (anti-UCP1, 1:1,000; anti-BAX, 1:1,000; anti-BCL2, 1:1,000; and anti-caspase-3, 1:1,000; Proteintech, Wuhan, China) at 4 °C overnight. The membranes were then washed with TBS with Triton (TBST) and incubated with the secondary antibody (1:1,000, Beyotime, Shanghai, China) at room temperature for 2 h. Finally, a bound antibody was detected using the Omni-ECL™ Femto Light Chemiluminescence kit (EpiZyme, Shanghai, China) on a chemiluminescence imager (Bio-Rad).

White adipose tissue samples were fixed overnight in 4% paraformaldehyde, after which they were sliced into 5-μm-thick sections. Cells grown on coverslips were fixed in 4% paraformaldehyde at room temperature and permeabilized with 0.1% Triton X-100 (Sigma, United States). For histologic examination, tissue slices were stained with hematoxylin. For immunofluorescence and immunohistochemistry, tissue slices or cells were incubated with blocking buffer (1% bovine serum albumin) for 1 h, and then with anti-UCP1 (Proteintech, China), anti-COX IV antibody (Proteintech, China), or anti-MFN2 antibody (Cell Signaling Technology, United States) at 4°C overnight. After that, the tissue slices or cells were incubated with fluorescein isothiocyanate (FITC)–conjugated secondary antibody (for immunofluorescence) or horseradish peroxidase–conjugated secondary antibody (for immunohistochemistry) at room temperature for 1 h. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Zhongshan Jinqiao, ZLI-9557). Immunofluorescence images were captured with an AV300-ASW confocal microscope (Olympus America Inc., Center Valley, PA, United States).

Total proteins were extracted from mouse iWAT and pVAT using a Total Protein Extraction kit (Applygen, Beijing, China), and the protein concentrations were measured with a BCA protein assay reagent kit (Applygen, Beijing, China). Approximately 30 μg of protein was separated on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA) through a wet transfer (BIO-RAD, California, USA). The membranes were incubated with the primary antibodies (anti-UCP1, Proteintech, Wuhan, China; anti-PGC1α, Proteintech, Wuhan, China; anti-ZAG, Santa Cruz, Dallas, USA; anti-β-actin, CST, Danvers, MA, USA; anti-GAPDH, CST, Danvers, MA, USA) at a 1:100 to 1:1,000 dilution at 4°C overnight, followed by incubation with a goat anti-mouse/rabbit secondary antibody at a 1:5,000 dilution for 1 h (ZSGB-BIO, Beijing, China). The specific protein bands were visualized by enhanced chemiluminescence (Applygen, Beijing, China). The protein bands were quantified by Quantity One software (Version 4.6.9, BIO-RAD, California, USA). The expression levels of target proteins in iWAT were normalized to that of β-actin, and the expression levels in pVAT were normalized to that of GAPDH.