Claudin 1

Western blotting was performed as previously described [25 (link)]. Lysis buffer with added protease and phosphatase inhibitors were used for extracting total protein from intestinal tissues in each group (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China). After grinding tissue, the supernatant protein was collected and quantified via the bicinchoninic acid method. PVDF membranes were incubated with specific primary antibodies, which included rabbit monoclonals against occludin (1:1000; Abclonal), claudin-1 (1:1000; Abclonal), COX-2 (1:1000; Abcam), caspase-3 (1:1000; CST), Bcl2 (1:1000; Abclonal), Bax (1:1000; Abclonal), HMGB1 (1:1000; NOVUS), Nrf2 (1:1000; NOVUS), HO-1 (1:1000; Abclonal), and mouse polyclonals against GAPDH (1:1000; Abcam) and β-actin (1:1000; Abcam) at 4 °C overnight. After incubation with the primaries, the membrane was washed three times, then incubated with goat anti-rabbit (1:5000; Zhongshan Golden Bridge, China) and goat anti-mouse (1:5000; Zhongshan Golden Bridge, China) IgG antibodies at room temperature for 1 h, and then washed three times. Lastly, the membranes incubated with enhanced chemiluminescence were exposed to the ChemiDoc Touch Imaging System. The grayscale values of protein bands were analyzed using Image J.

Frozen 6 μm thick tissue sections were prepared as described above [24 (link)]. Tissue sections were blocked by five percent bovine serum albumin in phosphate-buffered saline (PBS) with 0.1% Triton X-100, which were then incubated at 4 °C overnight with primary antibodies against matrix metalloproteinase-9 (MMP-9) (1:500, rabbit IgG; CST), MPO (1:500, rabbit IgG; CST, Danvers, MA, USA), occludin (1:200, rabbit IgG; Abclonal, Woburn, MA, USA), claudin-1 (1:200, rabbit IgG; Abclonal), COX-2 (1:500, rabbit IgG; Abcam, Cambridge, UK), 4-HNE (1:200, rabbit IgG; Abcam), and 8-OHDG (1:200, rabbit IgG; Santa Cruz, Dallas, TX, USA). After being rinsed four times with PBS for 5 min, the sections were then incubated with secondary antibody (1:500, Dylight488 goat anti-rabbit IgG, Abcam) for 1 h. After being washed three times with PBS, a mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) was added to the sections, which were then sealed with a coverslip. Fluorescent signals were observed under a fluorescence microscope (Olympus BX61, Tokyo, Japan). Three areas were selected from each slide for analysis, and mean values were calculated.

Brain tissue was taken, the tissue was lysed on ice with RIPA lysis buffer, and the lysed material was blown with 1 mL pipette to destroy the DNA. After centrifuge, 3μL supernant was collected for BCA assay to quantify, and the remaining samples were incubated at 95°C for 5–10 min for denaturation and stored at −20°C. Prepare 10% SDS-PAGE gel, add 40 g protein per well, after electrophoresis separation of protein, 200 mA constant current for 1 hour to transfer protein to PVDF membrane, then blocked with 5% skim milk for 1 hour at room temperate. Then the primary antibody was incubated overnight at 4°C,including ZO-1, claudin-1, claudin-5, Occludin, eNOS were all purchased from ABclonal (Wuhan, China), iNOS (Cat:189885-1-AP, Proteintech, USA) and the internal reference antibody β-actin (Cat:R1102-1, Huabio, Hangzhou, China). Then membrane was washed with TBST for three times, and incubated with secondary antibody at room temperature for 1 h, and then membrane was washed with TBST for three times. TBST was discarded, ECL luminescent solution was added, and developed in the protein imager. Image J was used to quantify the gray value of the band.

IPEC-J2 cells were cultured in a 12-well plate. When the cell density reached 70%, the cells were co-treated with or without LPS, AMG487, and 3-MA. After 12 h, the cells were collected and fixed with 4% paraformaldehyde at 37 °C for 20 min. Next, the cells were incubated with PBS containing 1% bovine serum albumin (BSA) and 1% Triton X-100 at 37 °C for 2 h and then with a 1:100 dilution of Claudin-1 (ABclonal, Wuhan, China), p65 (Proteintech, Wuhan, China), and LC3 (Proteintech, Wuhan, China) antibodies overnight at 4 °C. After washing three times, 30 min later, the cells were conjugated with Alexa Fluor 488 affinity goat anti-rabbit IgG (H + L) (Proteintech, Wuhan, China) at room temperature for 1 h, and, finally, a laser confocal microscopy system (LSM880, Zeiss, Germany) was used to observe the protein expression in the cells.

Jejunum tissues were homogenized at 4°C in RIPA lysis buffer which contained protease inhibitors (PMSF) (Beyotime, Shanghai, China), and concentrations were determined using a BCA assay (35 (link)). Samples were further diluted, and 5× SDS-PAGE loading buffer was added and boiled for 5 min. Equal amounts of protein (10 μg) were loaded onto 12% SDS-polyacrylamide denaturing gels before being transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with tris-buffered saline Tween (TBST) containing 5% non-fat milk powder for 1 h at room temperature, the membrane was incubated overnight with diluted primary antibodies against ZO-1 (1:1,000; ABclonal, Wuhan, China), occludin (1:1,000; Selleck Chemicals, USA), claudin-1 (1:1,000; ABclonal, China), TLR4 (1:500; Proteintech, Wuhan, China), MyD88 (1:500; Wanleibio, Shenyang, China), NF-κB (1:500; Wanleibio, China), NLRP3 (1:1,000; Wanleibio, China), ASC (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), caspase-1 (1:500; Wanleibio, China), IL-1β (1:500; Wanleibio, China), IL-18 (1:1,000; Wanleibio, China), and GAPDH (1:5,000; Proteintech, China). Electrochemiluminescence liquid (ECL) (Tanon, Shanghai, China) was used to detect the signal. ImageJ software was used to assess protein levels. The target protein levels were normalized to GAPDH, and the radioactivity was compared with the control group.

The cells or colonic tissues were fully lysed, and total protein was extracted, which was separated on SDS–PAGE gel and transferred to a membrane. The primary antibodies ZO-1 (#ab96587,Abcam, UK), E-cadherin (#3195S, Cell Signaling Technology, MA, United States), occludin (#ab216327,Abcam, UK), claudin-1 (#A2196,ABclonal, China), Anti-Carbonic Anhydrase 9(CA9) (#ab243660,Abcam, UK), HIF-1α (#ab228649,Abcam, UK) and Histon H₃ (#4499S, Cell Signaling Technology, MA, United States) were added after blocking nonspecific binding sites with a protein blocker at 4°C for 24 h. Then, they were washed with the Tris–Tween buffer, and secondary antibody was added. This was followed by incubating for 1 h and washing. Then, a chemical luminescent substrate (#A38555, Thermo Fisher Scientific, MA, United States) was added and visualized with a fluorescent chemiluminescence imager. Densitometric analysis of bands was performed using the ImageJ software (version 1.47, National Institutes of Health, NIH, United States).