Anti pparγ

PCL was purchased from Beijing Heyanling Pharmaceutical Development Co. Ltd. (Beijing, China) and had been identified as the Genuine Medicinal Herb by Professor Lei Yan in Shandong First Medical University and Shandong Academy of Medical Sciences. A BCIP/NBT Alkaline Phosphatase Color Development Kit, DAPI Staining Solution, Blocking Buffer for Immunol Staining, Antifade Mounting Medium, a bicinchoninic acid (BCA) protein assay kit, and penicillin-streptomycin were purchased from Beyotime (Shanghai, China). A ReverTra Ace® Qpcr RT Kit and Taq SYBR® Green Qpcr Premix were purchased from TOYOBO (Shanghai, China). β-Glycerophosphate, P-nitrophenyl phosphate, and ascorbic acid-2-phosphate were purchased from Sigma-Aldrich (St. Louis, MO, USA). TRIzol was obtained from Invitrogen (Carlsbad, CA, USA). The anti-GAPDH, anti-PPARγ, anti-AhR, and CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) antibodies were obtained from Proteintech Group (Wuhan, China). Alizarin red stain was obtained from Cyagen Biosciences (Guangzhou, China). The primers were synthesized by BGI (Shenzhen, China).

Western blot analysis was performed as described previously^{37 (link)}. Briefly, samples were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes and incubated overnight at 4 °C with rabbit polyclonal anti-OB (1:1000; Millipore), anti-PPARγ (1:1000; Proteintech), anti-FABP4 (1:500; Proteintech) or mouse polyclonal anti-β-actin (1:10,000; Abcam) antibodies. Membranes were then incubated for 1 h at room temperature with fluorescence-labeled anti-mouse or anti-rabbit IgGs (1:5000) prior to visualization with an Odyssey Imager (LI-COR, Odyssey, NE, USA). Formalin-fixed adipose tissues were sectioned at 4 μm intervals, blocked with 1% BSA, and incubated overnight at 4 °C with anti-OB, anti-PPARγ and anti-PABP4 (1:1000 dilution for each antibody).

BBR and BRB (≥98%) were purchased from Shanghai Yuanye Biological Technology Co., Ltd (Shanghai, China). Metformin (MET) (≥98%) and fenofibrate (FEN) (≥98%) were obtained from Shanghai Aladdin Biochemical Technology Co. Ltd and the National Institute for Food and Drug Control, respectively. The mouse insulin ELISA kit was purchased from ZCIBIO Technology Co. Ltd (Shanghai, China). The total cholesterol (TC), triglyceride (TG), high-density lipid-cholesterol (HDL-c), low-density lipid-cholesterol (LDL-c), alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN), and creatinine (CRE) kits were purchased from Nanjing Jiancheng Institute of Biological Engineering (Nanjing, China). Anti–GLUT2 antibody, the primary antibody used for Western blotting, was obtained from Abcam plc (Cambridge, United States). Anti-ACC1, anti-FAS, anti-GSK3β, and anti–p-GSK3β-Ser9 antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, United States). Anti-ATGL was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, United States). Anti-GK, anti-PPARα, anti-CPT1, anti-CD36, anti-PPARγ, anti-PEPCK, anti-G6Pase, and anti–β-actin were purchased from Proteintech Group, Inc. (Wuhan, China). A secondary antibody was purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd (Beijing, China).

The amount of 40-50 μg protein prepared from mouse myocardium or NMVCs was usedin a standard western blot analysis. The polyvinylidene fluoride (PVDF) membrane binding sample protein was incubated with a high affinity anti-Col1a1 antibody (1:1000), anti-Col3a1 antibody (1:1000), anti-α-SMA (1:2000) (Santa Cruz Biotechnology, USA), anti-EZH1 (1:1000), anti-EZH2 (1:1000), anti-PPAR-γ (1:1000) (Proteintech, Rosemont, IL, USA), anti-p-NF-κB P65 (1:1000), anti-NF-κB P65 (1:1000)(Cell SignalingTechnology, USA), respectively. An anti-GAPDH antibody (1:2000) (Santa CruzBiotechnology, USA) was used to detect the level of GAPDH as an internal control. Protein was visualized using the ECL Plus detection system (GE Healthcare, Waukesha, WI).

Extracts of a skin sample or HaCaT cells were prepared and then centrifuged at 12,000 × g to precipitate the insoluble materials. The supernatant was retained and the concentration of protein in the supernatant was measured by a BCA Protein Assay Kit (Beyotime, Jiangsu, China). A sample of supernatant was resolved by SDS-PAGE using a 12% gel. The protein bands in the gel were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with 5% skim milk, followed by overnight incubation with anti-Filaggrin, anti-Involucrin, anti-PPARα, anti-PPARγ, anti-LXR (Proteintech, Chicago, IL, United States), or anti-ABCA1 (Absin, Shanghai, China). After that, the blot was washed and incubated with the appropriate secondary antibody (Cell Signaling Technology, Beverly, MA, United States). Finally, the blot was subjected to a detection assay using a chemiluminescence substrate (Pierce, Rockford, IL, United States), and images of the blot were acquired using an Amersham Imager (GE Healthcare Biosciences, Pittsburgh, PA, United States).