Fig. 5.
Effects of C-CPE220 on tight-junction permeability in Caco-2 monolayer cells. A, interaction of C-CPE220 and claudin-4 in Caco-2 lysate. Ten micrograms of C-CPE or C-CPE220 was incubated with Caco-2 lysate (10 μg protein) for 30 min at 37°C. Ni-NTA resin was added, and then the mixture was incubated for an additional3hat 4°C. Then the samples were centrifuged, and the resultant supernatant fraction (Sup) was prepared. The precipitated resin was washed with the buffer for pull down. The resultant precipitated fraction containing resin (Ppt) was prepared. The Sup and Ppt fractions were subjected to SDS-PAGE followed by immunoblotting using anti-His-tagged antibody and anti-claudin-4 antibody. Data are representative of four independent experiments. B, interaction of C-CPEs and EC2hCld-4. The immunoplate was coated with GST or GST/EC2hCld-4 (10 μg/ml). After blocking the wells, the wells were treated with C-CPEs at the indicated concentration for 2 h at room temperature. The wells were washed, and anti-His-tagged Ab was added to the wells. After an additional 2 h of incubation at room temperature, the wells were washed and incubated with the peroxidase-labeled Ab for 2 h at room temperature. Finally, a substrate for peroxidase was added, and the resultant product was visualized. *, significantly different from the GST-coated values (p < 0.05). Data are means ± S.D. (n = 4). The data are representative of three independent experiments. C, effect of C-CPEs on tight-junction permeability in Caco-2 monolayers. Caco-2 cells were seeded onto Transwell. When the transepithelial resistance was stable, we added vehicle, C-CPEs, or 30-aa peptides to the basal side at the indicated concentration. TER values were monitored at 0 and 18 h after the addition of C-CPEs by an electrode. Data are representative of three independent experiments. *, significantly different from the value at 0 h (p < 0.05). Data are means ± S.E. (n = 4).