Cells or tissues were homogenized in ice-cold RIPA buffer with a proteinase inhibitor cocktail (Sigma-Aldrich). Protein concentrations were determined using the BCA Protein assay kit (Thermo Scientific). Equal amounts of protein were fractionated by 10% SDS polyacrylamide gels, followed by immunoblotting with the following primary antibodies: asprosin (FNab09797), UCP1 (ab10983; Abcam), PGC1a (ab54481; Abcam), β-tubulin (ab6046; Abcam). Membranes were then incubated with peroxidase-conjugated secondary antibody, and specific bands were detected with a Bio-Rad (Hercules, CA) imaging system.

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