Ecl luminescence reagent

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).

MKN1 cells were plated in 6-well dishes (3 × 10⁵ cells/well) overnight and then and incubated with compound KL-6 at different concentrations (0, 1, 2, or 5 µM) for 24 h. RIPA buffer containing an inhibitor of serine protease and phosphatase inhibitor was used to lyse the cells. The cell lysates were quantified, separated by an SDS-PAGE gel, and transferred to a PVDF blotting membrane (GE Healthcare, USA), which was further blocked by 5% milk and incubated with primary antibodies, including anti-GAPDH (#5174S), anti-STAT3 (#12640S), and anti-Phospho-Stat3 (Tyr705; # 9145S; Cell Signalling Technology, Shanghai, China) at 4 °C overnight. The membranes were washed with TBST and incubated with a second antibody, antirabbit (#7074) at room temperature for 2 h. The antibody-protein complexes were detected using an ECL luminescence reagent (Biosharp, Shanghai, China). The experiment was independently conducted at least three times.