Anti ucp1

As previously described, proteins were extracted from the kidney22 and from the aorta.23 Electrophoresis was performed with 4% to 15% acrylamide Criterion gels (Bio‐Rad, Hercules, CA). Protein transfer onto nitrocellulose membranes (Bio‐Rad) was performed with the Trans‐Blot Turbo Transfer System (Bio‐Rad). Blots were blocked with Tris‐buffered saline–Tween‐BSA 5% and then incubated with the following primary antibodies: anti‐eNOS (endothelial nitric oxide synthase; BD Biosciences, Franklin Lakes, NJ; #610297), anti–phosphorylated eNOS (pS1177, BD Biosciences, #612393), anti‐CD45 (Abcam, Cambridge, UK; ab10558), anti‐NRF1 (Cell Signaling Technology, Danvers, MA; 46743s), anti‐UCP1 (Cell Signaling Technology; #14670), anti‐OPA1 (BD Biosciences; #612606), anti‐FIS‐1 (Santa Cruz Biotechnology, Dallas, TX; sc98900), anti‐p47 (BD Biosciences; #610355), anti‐gp91 (BD Biosciences; #611414) anti‐COX2 (BD Biosciences; #610204), and anti–β‐actin (Sigma, St. Louis, MO; A5316). Following incubation with a goat antirabbit IgG (H+L) or a goat antimouse IgG (H+L) secondary antibody, and the horseradish peroxidase conjugate (Thermo Fisher Scientific, Waltham, MA), the reaction was developed by enhanced chemiluminescence (Bio‐Rad) according to the manufacturer's instructions, and the signal was visualized by chemiluminescence.

The liver and fat tissues of each mouse were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into slides with a thickness of 4 μm. Liver and fat tissue sections were stained with hematoxylin and eosin (H&E) for histological analysis. For oil red O staining, liver tissues from the same liver lobe were cut into small pieces, then the frozen sections were rinsed in distilled water and stained with 0.2% oil red O (Sigma) and 60% 2-propanol (Sigma) for 10 min at 37°C. For immunohistochemistry analysis, fat slides were rinsed in 0.01 mol/L sodium citrate (pH 6.0) and heated for 20 min in a microwave to retrieve antigen. The sections were blocked in blocking buffer containing 5% goat serum, 2% BSA, 0.1% Triton X-100 and 0.1% sodium azide in PBS, then incubated overnight with anti-UCP1 (Cell Signaling) by a dilution of 1:100 at 4°C. After being washed twice in PBS, slices were incubated with secondary antibodies (Cell Signaling) for 1 h at room temperature. Slides were counterstained with H&E. All digital pictures were acquired using an EVOS X1 microscopy (Thermo Fisher Scientific).

Liver and adipose tissues were fixed in 4% paraformaldehyde, then embedded in paraffin and cut into 4-μm thick slides. The slides were stained with haematoxylin and eosin (H&E) for histology analysis. For immunohistochemistry staining, slides were antigen retrieved by submerged in 0.01 mol/L sodium citrate (pH 6.0) and heated for 20 min in a microwave. The sections were blocked in blocking buffer containing 5% goat serum, 2% BSA, 0.1% triton X-100 and 0.1% sodium azide in PBS, then incubated overnight with anti-UCP1 (Cell Signalling Danvers, USA) by a dilution of 1:100 at 4 °C. After washed twice in PBS, slices were incubated with secondary antibodies (Cell Signalling, Danvers, MA, USA) for 1 h at room temperature. Slides were counterstained with H&E and digital images were collected with an EVOS X1 microscopy (Thermo Fisher Scientific, Shanghai, China).