Protein Extraction and Western Blot Protocol

Total Protein Extraction Kit was used for the extraction of total proteins of the ileum and colon, followed by determining the concentration of proteins by Standard BCA Protein Assay Kit. The underlined kits were procured from Keygen Biotech (Nanjing, China). Western blotting was used for evaluating the expression level of proteins [29 (link), 30 (link)]. Briefly, the separation of proteins (in equal amounts) was carried out by an SDS-PAGE, followed by transferring into a PVDF membrane (Millipore, USA). Then, skimmed milk (5%) was used for membrane blockage for 60 min at ~25°C and then overnight incubated with appropriate primary antibodies for iNOS (1 : 1000, Proteintech, China), FXR (1 : 4000, Abcam, USA), TGR5 (1 : 4000, Abcam, USA), and β-actin (1 : 5000, Abcam, USA) at 4°C. After washing with TBST, membranes were incubated with secondary antibodies (1 : 5000, EarthOx, USA) for 1 h at room temperature. Chemiluminescence detection was performed using an ECL luminescence reagent (Biosharp, Hangzhou, China) according to the manufacturer's instructions. Specific bands were detected, analyzed, and quantified by ImageJ software (NIH, Bethesda, MD, USA).