Figure 4.

Expression of kAE1wt increases paracellular permeability of filter-grown MDCK cells. Cells grown to polarity on Transwells were biotinylated at either the apical (AP) or basolateral (BL) surface and then lysed. Western blotting of the initial cell lysate (total lysate lanes are the loading control) and the isolated biotinylated fraction (surface fraction) shows that in untransfected cells, only trace amounts of the basolateral proteins E-Cadherin and Na⁺/K⁺-ATPase were detected at the apical surface, whereas proportions at both surfaces seemed equal in kAE1-expressing cells. A significant proportion of kAE1wt was also apparently localized to the apical surface. These biotinylation data are at odds with immunofluorescence images for all three markers in the same kAE1wt-expressing MDCK cells (Figures 3 and 6 show kAE1wt), which localize all three proteins to the basolateral membrane only, indicating that kAE1wt increases paracellular permeability to biotin. Representative of two separate experiments.