Rabbit anti mouse ucp1

For immunofluorescent staining, cells were fixed with 4% paraformaldehyde and blocked with 5% BSA for 2 h at room temperature, and then incubated with the primary antibody rabbit anti-mouse UCP1 (dilution, 1:200; Proteintech, Chicago, United States). After washing, sections were incubated with the secondary antibody Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin G (1:200; Thermo Fisher, Holtsville, NY, United States). Nuclei were stained with DAPI (Solarbio, Guangzhou, People’s Republic of China).

Whole beige adipocyte lysates (25 μg) in RIPA buffer containing protease inhibitor cocktail and phenylmethylsulfonyl fluoride were prepared using 5× SDS-PAGE loading buffer [250 mM Tris-HCl (pH 6.8), 0.25% bromophenol blue, 50% glycerol, 10% SDS, 0.5 M DTT (Bioseang Inc., Seongnam, Korea)] for separation by SDS-PAGE. After electrophoretic separation, gels were transferred onto activated polyvinylidene difluoride membranes for 1 h at 0.4 A. Membranes were blocked with 5% BSA for 2 h at room temperature and incubated with the primary antibody, rabbit anti-mouse UCP1 (dilution, 1:500; Proteintech, Chicago, United States)., which was diluted in blocking solution, for a minimum of 12 h at 4°C. Membranes were then incubated with secondary horseradish peroxidase-conjugated antibodies, HRP-conjugated affinipure goat anti-mouse immunoglobulin G (1:10,000; ZSGB-BIO, Inc., Beijing, People’s Republic of China)) for 2 h at room temperature, before visualization using the Supernova ECL western blotting detection system and imaging with the ChemiDocTMTouch (BioRad, CA, United States). Band intensities were quantified using ImageJ software. Assays were performed with samples from at least three independent experiments.