Occludin

Antibodies for p65, p-p65, BDNF, claudin-5, occludin, caludin-1, iNOS, COX-2, and β-actin were purchased from Cell Signaling Technology (Beverly, MA). Enzyme-linked immunosorbent assay (ELISA) kits for cytokines were purchased from Ebioscience (San Diego, CA). 4′,6-Diamidino-2-phenylindole dilactate (DAPI) was purchased from Sigma (St. Louis, MO). QIAamp Fast DNA stool mini kit was purchased from Qiagen (Hilden, Germany). Limulus amoebocyte lysate (LAL) assays was purchased from Cape Cod Inc. (East Falmouth, MA). General anaerobic medium (GAM), BL, and DHL media were from Nissui Pharmaceutical Co. (Tokyo, Japan). MRS medium was purchased from BD (Radnor, PA).

Whole cell lysates were prepared in RIPA buffer (Sigma) and quantified using Bradford Protein Assay (Bio-Rad). Twenty micrograms of lysates were resolved on Bolt^® 4–12% Bis-Tris Plus gels using Bolt^TM MOPS SDS Running buffer and transferred to a Hybond C super nitrocellulose membrane (GE Healthcare). After blocking to prevent non-specific binding in 5% milk in PBST for 1 h at room temperature, membranes were incubated with the specific primary antibodies overnight at 4 °C. The following primary antibodies were used: E-cadherin (Takara Bio Inc., M106, clone HECD-1, 1:1000 dilution), Occludin (Cell Signaling, #5446, 1:1000 dilution), Vimentin (Cell Signaling, #5741, 1:3000 dilution), ZEB1 (Santa Cruz, sc-25388, 1:500 dilution), SNAI1 (Santa Cruz, sc-28199, 1:500 dilution), LIN28B (Cell Signaling, #4196, 1:1000 dilution), LIN28A (Cell Signaling, #3978, 1:1000 dilution), SMAD2/3 (Cell Signaling, #8685, 1:1000 dilution), β-actin (Abcam, ab8227, 1:200,000 dilution), GAPDH (Santa Cruz, sc-137179, 1:10,000 dilution). Following incubation with the specific HRP-conjugated antibody (Dako, #P0447 or #P0448, 1:2,500 dilution), chemiluminescence signal was detected using Amersham^TM ECL^TM Western blotting detection reagents (GE Healthcare) and Amersham Hyperfilm^TM ECL (GE Healthcare). Uncropped scans of the most important blots are shown in Supplementary Fig. 13.

Colon samples were excised and homogenized in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing 1 mM Pierce™ phosphatase inhibitor (Thermo Scientific, CA, U.S.A.) and 0.1% Halt™ protease inhibitor cocktail (Thermo Scientific, CA, U.S.A.). The mixture was centrifuged (13000 rpm, 20 min, 4°C) and the supernatants were collected. Protein concentration was measured using Bradford’s colorimetric method. Next, the extractive was mixed with a 5× SDS/PAGE sample buffer. Equal amounts of proteins (30 μg) were separated by a 10% SDS/PAGE gel and then transferred to PVDF membranes (Merck Millipore). After blocking in a 5% BSA buffer for 1.5 h, membranes were incubated with respective primary antibodies (occludin, zonula occluden-1 (ZO-1), TNF-α, nuclear factor-κB (NFκB), IκB) (Cell Signaling Technology, MA, U.S.A.) at 4°C overnight. After washing with TBST three times, membranes were incubated with a secondary antibody (Bioworld, U.S.A.) for the target bands for 1.5 h at room temperature. For visualization of the bands, all membranes were incubated with the immobilon western chemiluminescent HRP substrate (Millipore, U.S.A.) for desired durations. The relative density of the band for the protein of interest was compared with the band for the housekeeping gene GAPDH in each group.