Luminal reagent

Total protein colon samples were extracted using RIPA buffer (P0013C, Beyotime, Shanghai, China), and 15 μg proteins were separated by 10% SDS-PAGE electrophoresis and transferred to a methanol-activated PVDF membrane (GVPPEAC12, Millipore, Bellerica, USA). The membrane was blocked for 1 h in PBST containing 5% milk and subsequently probed with Occludin antibody (GTX114949, GeneTex, Texas, USA), ZO-1 antibody (Ab96587, Cambridge, UK), P65 antibody (YT3108, ImmunoWay, Texas, USA), phosphorylated P65 antibody (B4568, ImmunoWay, Texas, USA), and GAPDH antibody (ab9485, Abcam, Cambridge, UK), respectively, for 2 h. After 1 h incubation with goat-anti-rabbit HRP-conjugated secondary antibody (ab97051, Abcam, Cambridge, UK), the protein bands were detected with luminal reagent (Millipore, Pittsburgh, USA) and their relative intensities were quantified using Image J. Biological verifications were performed three times in each group.

After induction for 16 h, 1 ml of culture was harvested by centrifugation at 2000g, 4 °C for 10 min and re-suspended in 200 µl of PBS. For 200 µl of cells, 4 µl of 10 mg/ml trypsin (Merck, 108367) was added. After incubation at 37 °C for 10 min, the cells were put on ice immediately and 20 µl of FBS (fetal bovine serum) was added to stop digestion. The cells were harvested, washed three times with PBS containing 10 % FBS and once in PBS alone. The washed cells were finally re-suspended in 5 % SDS loading buffer and boiled for 20 min. As a control, another 200 µl aliquot of cells was simultaneously processed in the same manner without added trypsin. Samples were resolved by SDS/PAGE (12 % gels) and transferred on to nitrocellulose membranes (Millipore) for 70 min at 300 mA at 4 °C. The non-secreted and secreted proteins were detected by anti-His antibody (Sigma) and horseradish peroxidase-conjugated goat anti-mouse secondary antibody (ZSGB-Bio) with a working dilution of 1:3000 and 1:5000 respectively. The membranes were incubated in luminal reagent (Millipore) and exposed to film.