Goat anti mouse

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).

After 48 h of transfection, the DPCs proteins were disposed of with RIPA lysis buffer (Beyotime, Shanghai, China), and concentrations were detected using the BCA method. The proteins were separated and then transferred to PVDF membranes, which were probed with 1:500 rabbit anti-EGR1 (Affinity, Melbourne, Australia), 1:2500 mouse anti-GAPDH (ABclonal, Wuhan, China), 1:1000 rabbit anti-PCNA (Abcam, Cambridge, UK), 1:1000 rabbit anti-CDK2 (Abcam, Cambridge, UK), 1:3000 goat anti-rabbit IgG HRG antibody (ABclon, Wuhan, China), and 1:3000 goat anti-mouse (ABclonal, Wuhan, China). The protein expressions were measured using the ECL Western Blot kit (BioSharp, Hefei, China), and analysed by the ChemiDoc^TM Analysis System (Bio-Rad, Hercules, CA, USA).