Ab167161

A BCA assay was used to measure the concentration of total protein extracted from homogenized duodenum tissues. Western blot was performed following standard procedures. Briefly, 20μl denatured protein each well was loaded onto SDS-PAGE. After electrophoresis, the proteins were electroblotted to a PVDF membrane. Incubations of the primary and secondary antibodies were performed overnight at 4°C and for one hour at room temperature, respectively. Anti-Tight Junction Protein 1 (ZO-1) antibody (NBP1-85047) was purchased from Novus (Shanghai, China). Anti-Occludin (OCLN) (ab167161), anti-Claudin 3 (CLN3) (ab15102), anti-Desmocollin 2 (DSC2) (ab230039), anti-Toll-like receptor 4 (TLR4) (ab217274), anti-IKK alpha + IKK beta (ab178870), anti-NK-κB p65 (ab16502) and anti-beta Actin (ab8227) antibodies were purchased from Abcam (Shanghai, China). After washing with TBST three times for 10 min, ECL detection, exposure and development were performed. Protein band intensity was measured and quantified by Image Lab software.

Relative protein levels of CuSOD, MnSOD, GPX1, GPX4, ZO-1, Claudin-1, Occludin, IL-2, IL-8, and IL-10 in the jejunum were determined using western blotting. Colon samples were collected and the protein expression of CuSOD, MnSOD, GPX1, GPX4, IL-2, IL-8 and IL-10 was determined [22 (link)]. The resultant signals were obtained using Quantity One software (Bio-Rad, Hercules, CA, USA). Primary antibodies were used as follows: CuSOD (1:50000; ab51254, Rabbit, Abcam, UK), MnSOD (1:1000; ab68155, Rabbit, Abcam, UK), GPX1 (1:1000; bs-3882R, Rabbit, Bioss, Beijing, China), GPX4 (1:1000; 14432-1-AP, Rabbit, Proteintech, Rosemont, IL, USA), ZO-1 (1:3000; 21773-1-AP, Rabbit, Proteintech, USA), Occludin (1:1000; ab167161, Rabbit, Abcam, UK), Claudin1 (1:500; ab15098, Rabbit, Abcam, UK), IL-2 (1:2000; ab92381, Rabbit, Abcam, UK), IL-8 (1:2000; ab110727, Rabbit, Abcam, UK), IL-10 (1:1000; 20850-1-AP, Rabbit, Proteintech, USA) and Actin (1:5000; 66009-1-Ig, Mouse, Proteintech, USA).

The expression and distribution of ruminal epithelium tight junction proteins were performed via immunohistochemistry. The following antibodies were used in the immunohistochemistry analysis: claudin-1 (ab211737; Abcam), claudin-4 (ab210796; Abcam), occludin (ab167161, Abcam), and ZO-1 (ab214228, Abcam). Specimens were embedded in paraffin after having been fixed via 4% paraformaldehyde solution, sectioned and incubated with antibodies, and dyed by hematein for light microscope observation. Image Pro Plus v.6.0 software was used to choose the same brown color as the consistent criterion for estimating all photos. Each photo was determined to acquire the cumulative optical density of each image.

Western blot analyses were performed as described previously [23 (link)]. Primary antibodies against inducible nitrogen oxide synthase (iNOS) (Abcam, ab178945, Cambridge, UK), cyclooxygenase 2 (COX-2) (Abcam, ab179800, Cambridge, UK), zonula occludens-1 (ZO-1) (Abcam, ab96587, Cambridge, UK), occludin (Abcam, ab167161, Cambridge, UK), signal transducer and activator of transcript 3 (STAT-3) (Abcam, ab68153, Cambridge, UK), p-STAT-3 (Abcam, ab32143, Cambridge, UK), and GADPH (Abcam, ab8245, Cambridge, UK) were used. Protein signals were detected with efficient chemiluminescence (ECL) reagent based on the manufacturer’s instructions.

Proteins were extracted from the proximal colon tissues and analyzed on Ready Gel Tris-HCl (Bio-Rad). The tissues were homogenized in RIPA buffer (1% IGEPAL, 0.5% sodium deoxycholate, and 0.1% SDS in Tris-buffered saline solution [pH 7.4]), supplemented with protease inhibitor cocktail (Sigma-Aldrich). The homogenate was centrifuged at 14,000g for 10 minutes at 4°C. Sample lysates were denatured at 95°C for 5 minutes in the presence of 4× LDS sample loading buffer (Invitrogen) and 5% β-mercaptoethanol (Bio-Rad). Equal amounts of protein (30 μg) were separated by 4%–20% Ready Gel Tris-HCl gels (Bio-Rad), transferred to polyvinylidene difluoride membranes, and blocked with StartingBlockT20 blocking buffer (Thermo Fisher Scientific) for 60 minutes at room temperature. Membranes were incubated with rat anti–ZO-1 monoclonal antibody (MABT 11, Sigma-Aldrich) and rabbit recombinant monoclonal anti-OCLN antibody (ab167161, Abcam) at 1:400 dilution at 4°C overnight, and they were then washed in Tris-buffered saline for 1 hour. The membranes were then probed with peroxidase-conjugated secondary antibodies at 1:8000 dilution for 1 hour at room temperature, and the bands were visualized by electrochemiluminescence (ECL, Thermo Fisher Scientific). Signals were quantified using ImageJ (NIH) and normalized to controls.