Goat anti rabbit

The hypothalamus and distal ileum were collected 8 weeks postsurgery and lysed in radioimmunoprecipitation assay buffer. Protein concentrations of the supernatant were quantified using the BCA Protein Assay Kit (Solarbio Life Science, Beijing, P. R. China), and equivalent amounts of protein were subjected to 10% sodium dodecyl sulfate−polyacrylamide gel electrophoresis and transferred to a 0.2 μm aperture polyvinylidene fluoride membrane. The membranes were blocked in 5% bovine serum albumin liquid and incubated with primary TGR5 (Abcam, Cambridge, UK), FXR (ABclonal, Wuhan, P.R. China), MC4R (ABclonal, Wuhan, P.R. China), Klotho (Proteintech, Wuhan, P.R. China), POMC (Wuhan, P.R. China) and β-actin (Cell Signaling Technology, Danvers, MA, USA) antibodies at 4 °C overnight. The membranes were washed three times with Tris-buffered saline and Tween 20, goat anti-rabbit (ABclonal, Wuhan, P.R. China) and goat anti-mouse (ABclonal, Wuhan, P.R. China) were incubated at room temperature for 1 hour with shaking. After three rinses with TBST solution, the membrane was scanned. The relative concentration of protein was quantified by densitometry using the Tanon Imaging System and ImageJ software.

The paraffin section of the kidney was deparaffinized and rehydrated using xylene and alcohol in gradients. Next, the sections were heated using citrate buffer pH 6 for 20 min for antigen retrieval and followed by blocking non-specific antigen using blocking serum for 20 min. Then, the slides were incubated with primary antibodies, PDGFR-β (1:200 dilution, Abclonal, Cat. No. A19531) and α-SMA (1:400 dilution, Sigma, Cat. No. A2547), overnight. On the following day, the slides were incubated with secondary antibody, goat-anti-mouse (Abclonal, Cat, No. AS076), and goat-anti-rabbit (Abclonal, Cat. No. AS039) for 1 h, and DAPI staining for 20 min. Finally, the slides were observed under a confocal microscope (Zeiss, Cat. No. LSM900).

SDS-PAGE and assays were performed using routine methods. The cells were treated in RIPA lysis buffer, whereafter the lysed cell protein was centrifugated. The separated proteins were analyzed through 15% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Darmstadt, Germany). The membrane was blocked with 2% BSA (Vazyme, China) and incubated with the rabbit anti-His antibody (1:1000, ABclonal, Wuhan, China). The target proteins were visualized with chemical luminescence substrate with Amersham Imager 600 (GE, Atlanta, GA, USA) after incubation with HRP conjugated secondary antibodies goat anti-rabbit (ABclonal, China, 1:2000 dilution).