Proteins were extracted from cells or tissues as previously described [34 (link)]. Equal amounts of cellular proteins, 30 to 50 µg, were separated by electrophoresis using gradient gels (4–15%) and blotted onto PVDF membranes. Following blocking, membranes were incubated. Primary antibody incubation was performed overnight at 4 °C (anti-UCP1, Abcam #ab10983, dilution 1:1000; anti-TBP, CST #D5C9H, dilution 1:1000). Primary antibodies were detected with HRP-conjugated anti-rabbit or anti-mouse immunoglobulins (Promega, Charbonniere Les Bains, France). Detection was performed using Immobilon Western Chemiluminescent HRP Substrate (Merk-Millipore, Fontenay Sous Bois, France). Chemiluminescence obtained after adding Pierce ECL Western blotting substrate (Thermo Scientific, Asnièrse sur Seine, France) was detected using an Amersham Imager 600 and quantified with Image Lab 5.0 software (Bio-Rad, Marnes-la-Coquette, France).
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