Figure 5.

rEhCP112a enters to Caco-2 cell monolayers through the intercellular space. (A) rEhCP112p coupled to Alexa 647 (red) was apically added to Caco-2 cell monolayers for different times, then, cells were fixed and analyzed through confocal microscope in the xy- and zy-planes. Square areas are magnified in (B). (C) Caco-2 cells treated as in (A) were incubated with α-occludin antibody. (D) As in (A), but rEhCP112p was pre-incubated with E-64. Labeled papain was used as control. Nuclei (n) were counterstained with DAPI (blue). Arrows: labeled enzymes. Bar: 10 μm. (E) WB assays of recovered supernatants (SN) from Caco-2 cells treated with refolding buffer or rEhCP112p or rEhCP112p-E-64. Input: enzymes added to epithelial cells. CL, cell lysates.