Anti adrb3

The liver and eWAT were excised from each mouse, fixed in 10% (v/v) paraformaldehyde in phosphate buffered saline (PBS), and embedded in paraffin for staining with hematoxylin and eosin (H & E) and Masson’s trichrome dye. The stained slices were examined under an optical microscope (Zeiss Axioscope) at 200× magnification [31 (link)]. For immunohistochemistry, paraffin-embedded sections were incubated at 4 °C with anti-ADRB3, anti-ATP5L, anti-PRKAG3, or anti-UCP3 (Abcam, Cambridge, MA, USA), followed by detection using the ABC Vectastain-Elite kit (Vectastain ABC kit, Vector Labs, Burlingame, CA, USA) according to the manufacturer’s instructions.

Cells were washed two times with cold PBS and lysed in RIPA buffer (0.01 M Tris, pH 7.4, 0.15 M NaCl, 0.001 M EDTA, 0.001 M EGTA, 1% Triton-X 100, 0.002 M PMSF, and protease inhibitor (Roche, Rotkreuz, Switzerland)). After centrifugation of lysate, supernatant was used for immunoblot assay. Protein concentration was measured by BCA protein assay system (Pierce Biotechnology, IL, USA). Equal amounts of protein from each sample were separated by SDS-PAGE. Anti-Adrb3 (Abcam, Cambridge, UK, #ab59685), anti-UCP-1 (EMD Milipore, CA, USA, #AB1426), anti-pAKT (Cell signaling, #9271 S), anti-AKT (Cell signaling, #9272), anti-Hsc70 (Rockland, PA, USA, #200-301-A28), and anti-TonEBP antibody^{5 (link)} were used for immunoblotting at 1:1000 dilution. HRP-conjugated mouse, rabbit, or goat secondary antibodies were used for detection. The antigen–antibody binding was detected by chemiluminescent detection reagent (GE Healthcare, NJ, USA). Uncropped western blots are shown in Supplementary Fig. 12.