Ab276131

The cells were lysed for 20 min in 180 μL of lysis buffer (Beyotime, Shanghai, China) on ice before the centrifugation at 4 °C and 14,000 rpm for 10 min. The total protein concentration was estimated using a bicinchoninic acid (BCA) protein detection kit (CWBIO, Shanghai, China) with bovine serum albumin (BSA) as the standard. The proteins were separated by sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After being blocked with 5% (w/v) skimmed milk for 2 h, the membranes were probed with corresponding primary antibodies overnight at 4 °C and then incubated with secondary antibodies conjugated with horseradish peroxidase for 2 h. The primary antibodies used were: TLR4 (1:1000, Abcam, ab13556), myeloid differentiation factor 88 (MyD88, 1:1000, Abcam, ab133739), nuclear factor kappa-B (NF-κB) p65 (1:1000, Abcam, ab32536), NF-κB p65 (phospho S536) (1:1000, Abcam, ab76302), claudin-1 (1:2000, Abcam, ab211737), occludin (1:1000, Abcam, ab216327), ZO-1 (1:1000, Abcam, ab276131), myosin light-chain kinase (MLCK, 1:1000, ABclonal, A3835), and β-actin (1:5000, Abcam, ab179467). The protein intensity was quantified using the Image J software (version 1.8.0, NIH, Bethesda, MD, USA).

Colon tissue sections (4 mm) were deparaffinized in xylene twice for 10 min, rehydrated in a graded ethanol series once for 5 min, incubated in sodium citrate buffer for 10 min for antigen retrieval, blocked with 10% bovine serum albumin for 1 h, and incubated with antibodies against Claudin-1 (1:100; Abcam, Cambridge, UK; ab242370), zonula occludens 1 (ZO-1) (1:100; Abcam; ab276131), Occludin (1:100; Abcam; ab216327), mucin 2 (MUC2) (1:500; Abcam; ab272692), myeloperoxidase (MPO) (1:500; Abcam; ab208670), CD11b (1:100; Abcam; ab133357), IL-17 (2 mg/mL; Abcam; ab79056), IL-10 (10 mg/mL; Abcam; ab189392) overnight, and IL-1β (1:500; Abcam: ab283818). The sections were then incubated with secondary antibodies (SP9000; Zsbio Biotechnology, Beijing, China) for 1 h. The images were acquired using a BX53F microscope (Olympus, Tokyo, Japan).

The tissue sections were dewaxed, the antigens were repaired with citric acid, and 3% hydrogen peroxide was added to block the endogenous peroxidase. Afterward, 10% goat serum was added to block the tissue sections for a 30-min period, followed by overnight incubation using a primary antibody against MUC2 (ab27692, Abcam, Cambridge, UK) and ZO1 (ab276131, Abcam, Cambridge, UK) at 4 °C, after which they were incubated using secondary antibody (K5007, DAKO, Copenhagen, Denmark) for 50 min at an ambient temperature. Color development was performed with 3,3′-diaminobenzidine (DAB), and then they were stained with hematoxylin, dehydrated, and sealed for microscopic examination.