Immunoblot Analysis of UCP1 and UCP2 Expression

Immunoblot analysis was performed as previously described^{21 (link)}. Briefly, total proteins were extracted with RIPA buffer [50 mM Tris–HCl (pH 8.0), 0.15 M sodium chloride, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 1% NP-40] and both protease and phosphatase inhibitor cocktails (FUJIFILM Wako). The proteins were subjected to 10% SDS-PAGE and transferred to a PVDF membrane (ThermoFisher Scientific). The membranes were blocked with 3% skim milk followed by incubation with UCP1 antibody (MAB6158, R&D Systems, MN, USA), UCP2 antibodies (AF4739, R&D Systems) or β-Actin antibody (A5316, Sigma-Aldrich) at 4 °C overnight. The membranes were incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. Immunoreactive bands were detected by Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Darmstadt, Germany). Each band intensity was quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, USA). For evaluation of protein stability, 10 µg/ml Cycloheximide was treated for the time indicated before harvest. Full-length western blots are shown in Supplementary Fig. S7.