Fig. 4.

(A) Zonulin at increasing concentrations was incubated on serum-starved Caco-2 cells. The cells were lysed, immunoprecipitated using anti-EGFR Ab, and processed for WB using anti-phospho EGFR (PY-Plus) Ab. To ensure equal loading, the blots were stripped and reprobed for EGFR. Zonulin caused a dose-dependent increase in EGFR phosphorylation that reached a plateau at 15 μg/mL. (B) Zonulin at 50 μg/mL was incubated either alone (lane 2) or in the presence of 5 μM of the EGFR-selective PTK inhibitor AG1478 (lane 3) on serum-starved Caco-2 cells. Cells exposed to media (lane 1) or AG1478 alone (lane 4) were used as additional controls. Zonulin caused an increase in EGFR phosphorylation that was completely abolished by the PTK inhibitor AG1478 (n = 3 experiments). (C) Zonulin, either alone or in the presence of 5 μM AG1478, was applied to the luminal side of C57BL/6 WT intestinal segments at a concentration of 50 μg/mL, and TEER was measured at baseline (open bars) and 90 min postincubation (closed bars). Zonulin caused a significant drop in TEER that was prevented by the presence of AG1478 (n = 4 mice for each group). t₀, time point t = 0. (D) The zonulin-induced EGFR phosphorylation was significantly reduced following treatment with 2-chain mature HP2 (50 μg/mL; lane 3) compared with single-chain zonulin (lane 2). Lane 1 shows EGFR phosphorylation in cells treated with media alone.