Zo 1 21773 1 ap

The tissues were lysed using RIPA lysis buffer (Beyotime Tech, Shanghai, China) with 1% PMSF (Beyotime Tech, Shanghai, China) and phosphatase inhibitor cocktail (BosterBio, Nanjing, China) to extract total protein. The concentration of proteins was determined by using a BCA assay (KeyGen Biotech, Nanjing, China). Tissue protein (30 μg/lane) was electrophoresed through SDS-PAGE at 90 V for 15 min and 120 V for 1 h, and then, electronically transferred to PVDF membranes at 260 mA for 1 h. After blocking with 5% de-fat milk for 2 h, the blots were incubated at 4 °C overnight with primary antibodies: β-actin (13E5) (purchased from Cell Signaling Technology (Boston, USA); ZO-1(21773-1-AP), occludin (27260-1-Ap), caspase-3 (66470-2-Ig), and JAK1 (66466-1-Ig) (purchased from Proteintech (Wuhan, China)); NF-κB p65 (ab32536), STAT3 (ab68153), and Nrf2 (ab76036) (purchased from Abcam (ab205718 and ab205719, Cambridge, UK)). After washing with TBST buffer three times, the HRP-conjugated secondary antibodies (Abcam, Cambridge, UK) were applied for 2 h at room temperature. Washing was performed again, and then the chemiluminescence signals were detected by using an ECL system (Biosharp, Hefei, China) and visualized by using a Chemiluminescence imager (Bio-Rad, California, USA). Finally, the images were analyzed using the Image Lab software. All expression levels were normalized to β-actin.

Tissues were extracted using RIPA lysis buffer with cOmplete™ protease inhibitor cocktail (4693132001, Roche, Germany). The protein concentration was measured using the Pierce™ BCA protein assay kit (23227, Thermo Fisher) according to the manufacturer’s instructions. An equivalent amount of protein (40 μg) was separated on the SDS-PAGE gel and then transferred onto the 0.45 μM PVDF membranes (Millipore, Burlington, United States) according to the standard protocols. The membranes were blocked in 5% milk with TBST buffer for 2 h at the room temperature, followed by incubation with primary antibodies (1:1000, caspase-3, 9662, CST; 1:1000, Bax, 2772, CST; 1:1000, MyD88, 4283, CST; 1:1000, NF-κB, 8242, CST; 1:1000, GAPDH, 2118, CST; 1:1000, Claudin-1, ab15089, Abcam; 1:1000,TLR5, ab62460, Abcam; 1:1000, Bcl-2, ab194583, Abcam; 1:2000, β-actin, ab8227, Abcam; 1:2000, ZO-1, 21773-1-AP, Proteintech) at 4°C overnight. After incubation with a secondary antibody for 1 h at room temperature, the proteins were detected using an ECL reagent (Millipore, Burlington, United States).

Colon tissues were lysed in RIPA lysis buffer with 1% phosphatase inhibitor cocktail and 1% protease inhibitor cocktail (MedChemExpress, New Jersey, United States), they were homogenized and centrifuged in 4°C. Protein concentrations were measured with a Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, Massachusetts, United States). Electrophoresis of 40 μg protein was proceeded by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, United States). The membranes were blocked with 5% non-fat milk and incubated with antibodies. The antibodies are as follow: ZO1 (21773-1-AP, 1:1000, PROTEINTECH GROUP), MMP9 (ab76003, 1:3000, Abcam, Cambridge, MA, United States), GAPDH (ab8245, 1:5000, Abcam, Cambridge, MA, United States), HRP-conjugated goat anti-rabbit IgG secondary antibodies (SA00001-2, 1:5000, PROTEINTECH GROUP). The membranes were visualized using an ECL Plus kit (Thermo Scientific, United States) and exposed to MiniChem610 (SAGECREATION, Beijing, China). Images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, United States).