Figure 4

Immunofluorescence staining to localize tight and adherens junction proteins after Caco-2 cell monolayers were incubated with IIL ESPs treated with various single inhibitors. Caco-2 cell monolayers were incubated at 37 ℃ for 2 h with IIL ESPs pre-treated using various individual inhibitors (1.25 mM PMSF, 24 μM E-64, 2.4 mM 1,10-Phe or 10 μM pepstatin). Normal Caco-2 cells treated with 0.1% DMSO were used as a negative control, and 20 μg/mL ESPs without inhibitors was used to assess the hydrolysis of TJ proteins. The cells were fixed with 4% paraformaldehyde, permeabilized for 10 min with 0.25% Triton X-100 and blocked with 1% BSA. The cells were probed with primary antibodies against occludin, claudin-1, claudin-2 or E-cad and then incubated with FITC- or Cy3-conjugated secondary antibodies. Cell nuclei were stained blue with DAPI before being observed under a fluorescence microscope (1000×). Scale bars: 50 μm.