*4.6. Western Blotting*

To prepare tissue lysates, BAT and subWAT were homogenized on ice in ice-cold lysis buffer consisting of 25 mM Tris-HCl, pH 7.4, 10 mM sodium orthovanadate, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM EGTA, 1 mM phenylmethylsulfonyl fluoride and 1% NP-40 that supplemented with a protease inhibitor cocktail and phosphatase inhibitor cocktail (both, Roche Diagnostics, Mannheim, Germany). After centrifugation of the tissue homogenates at 14,000× *g* for 10 min at 4 ◦C, the supernatants were collected for determination of protein concentrations by Bradford protein assay using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins (5 μg for BAT or 25 μg for subWAT) were separated by SDS-PAGE (7.5% gel) and then transferred electrophoretically onto Clear Blot Membrane-P (ATTO, Tokyo, Japan) and immunoblotted with specific primary antibodies: UCP1 (ab10983, 1:2000 dilution; Abcam,) and β-actin (C4) (sc-47778, 1:5000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The secondary antibodies included goat anti-rabbit IgG (sc-2005, 1:8000 dilution) and m-IgGκ BP-HRP (sc-516102, 1:6000 dilution; both from Santa Cruz

Biotechnology, Inc.). ECL detection reagents (Amersham Biosciences, Buckinghamshire, UK) were used to detect the desired proteins, which were then quantified with the NIH Image software program (NIH, Bethesda, MD, USA).
