Gtx100674

Total protein was extracted by lysing cells with RIPA lysate I (C5000050010; Sangon Biotech, Shanghai, China). Protein concentration was measured using a BCA Protein Assay Kit (C5030210500; Sangon Biotech, China). Equal amounts of protein lysate were boiled for 5 minutes with 5× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (P1040; Solarbio, Beijing, China) and separated by SDS-PAGE Preparation kit (C631100; Sangon Biotech, China) and transferred to polyvinylidene fluoride membrane (Millipore ImmobilonTM P, Billerica, MA, USA). After blocking in 5% skim milk dissolved in Trisbuffered saline (0.02 M Tris base, 0.14 M NaCl, pH 7.4) containing 0.1% Tween 20, the membrane was incubated with primary antibodies overnight at 4°C, and then with IRDye 800CW goat anti-rabbit immunoglobulin G (Li-COR Biotechnology, Lincoln, NE, USA) for 1 hour at room temperature. The primary antibodies used in this study were UCP1 (ab10983; Abcam, USA), myf5 (ab139523; Abcam, USA), PPARγ (bs4590R; Bioss, China), CEBPα (GTX100674; Gene Tex, USA), β-actin (bs0061R; Bioss, China) and glyceraldehyde-3-phosphate dehydrogenase (HC301; TransGen, Beijing, China). The blots were imaged by the Odyssey CLx Imaging System (Li-COR Biotechnology, USA) and the protein density analysis was performed using Image J (v1.45) software.

To detect the expressions of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding protein-α (C/EBPα), and UCP1 in perirenal fat, immunohistochemical staining was performed on 5 μm thick tissue sections using rabbit polyclonal antibodies: PPARγ (bs4590R, BIOSS, Bengjing, China), C/EBP alpha (GTX100674, Gene Tex, Alton Pkwy, CA, USA), UCP1 (ab10983, Abcam, Waltham, MA, USA) as the first antibody in phosphate buffered saline-Tween. The secondary antibody was a horseradish peroxidase (HRP) (ab205718, Abcam, USA). Thermally induced antigen retrieval was performed by incubation with Tris-ethylene diamine tetraacetic acid for 20 minutes. Endogenous peroxidase was blocked by incubation with 3% H₂O₂ for 10 minutes. Non-specific binding was blocked by incubating sections in normal goat serum for 2 hours at room temperature. Visualization was performed by incubation with HRP conjugated streptavidin followed by staining with diaminobenzidine substrate. Images were analyzed using a Leica microscope (Leica, DM750, Wetzlar, Germany) equipped with a ICC50 W camera (Leica) and Leica Application Suite software (version 4.6.2) at a magnification of 400×. Image J (v1.45) software was used to determine the area of the positive signal divided by the total tissue area to calculate the area fraction of the positive signal.