Ab53032

After treatment, the mice in the study groups were sacrificed via euthanasia. The colonic tissues were dissected, isolated and washed with cold PBS. Then the samples were fixed with 4% paraformaldehyde. Embedded in paraffin, the samples were sliced into 5 μm sections. The sections were heated in the PBS in a microwave. After cooling down, the serum blocking solution was added to block the sections for half an hour at 37 °C. Then the sections were incubated with anti-Claudin-2 antibody(1:1000, #ab53032, Abcam, England) at 4 °C overnight and secondary antibody (#ab205718, Abcam) at 37 °C for one hour. After being washed with PBS, the sections were stained with diaminobenzidine for five minutes and hematoxylin for two minutes. Finally, the sections were observed under the microscope (Olympus Corporation).

Liver and intestinal samples were homogenized in RIPA buffer added with protease and phosphatase inhibitors. Full centrifugation at low temperature (15 min at 12,000 g) to obtain supernatant, and the protein concentration was quantified by BCA kit (Epizyme, Shanghai, China), proteins electrophoresis using the 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto 0.45 μm PVDF membranes (Millipore, United States). Subsequently, the PVDF membranes were socked in 5% skim milk containing 140 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.5), and 0.1% Tween 20°at room temperature for 60 min, and incubated with primary antibodies at 4°C overnight: FXR mouse monoclonal antibody (72105S, CST, United States), TGR5 rabbitpolyclonal antibody (72,608, Abcam, United States), ZO-1 (ab216880, Abcam, United States), Occludin (ab 216,327, Abcam, United States), Claudin 2 (ab53032, Abcam, United States), P-P65 rabbit monoclonal antibody (3031S, CST, United States), P65 rabbit monoclonal antibody (8242S, CST, United States), β-actin (Hua-an Biotech Inc., Hangzhou, China), and then incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for another 60 min. The protein bands were visualized by an ECL chemiluminescence detection kit (WBKLS0500, Millipore, United States) with an enhanced chemiluminescence system (Tanon 5200, Shanghai, China).

The total proteins of intestinal tissues were extracted by radioimmunoprecipitation assay protein lysis buffer (Invitrogen; Thermo Fisher Scientific, Inc., USA). A bisquinolinecarboxylic acid protein kit was used to measure the concentration of proteins. Then, 40 μg of protein from each sample was separated by 10% sodium salt-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane by the electric transfer method. Five percent skimmed milk powder was used blocking for 1 h. After incubation with primary and secondary antibodies, the protein bands were visualized by enhanced chemiluminescence. The primary antibodies were diluted at 1 : 1000 as follows: anti-LC3B (ab48394; Abcam, UK), anti-beclin 1 (ab210498; Abcam, UK), anti-p62 (ab109012; Abcam, UK), anti-ZO-1 (ab96587; Abcam, UK), anti-occludin (ab216327; Abcam, UK), anti-claudin-1 (ab180158; Abcam, UK), anti-claudin-2 (ab53032; Abcam, UK), anti-claudin-3 (ab52231; Abcam, UK), anti-Bcl-2 (ab182858; Abcam, UK), anti-Bax (ab32503; Abcam, UK), anti-cleaved Caspase-3 (ab49822; Abcam, UK), and anti-β-actin (ab8226; Abcam, UK). The grayscale value was analyzed by ImageJ.