Figure 5

Defective fence function of tight junction induced by COM crystals. (a) Proteins derived from whole cell lysate, apical membrane fraction and basolateral membrane fraction of controlled and COM-treated cells were subjected to western blot analysis using mouse monoclonal anti-Na⁺/K⁺ATPase-α1 as primary antibody. Each lane was loaded with proteins derived from equal number of cells (3.0 × 10⁵ cells per lane). Translocation of Na⁺/K⁺ATPase-α1 (a marker of basolateral membrane) from basolateral to apical membrane compartment was observed in the COM-treated cells, but not in the control cells. (b) The polarized MDCK cells were incubated with 100 μg/ml COM crystals for 48 h and then processed for immunofluorescence staining of Na⁺/K⁺-ATPase-α1 (in green) and nucleus (in blue). The COM crystals were detected in red by autoreflection at λ633 nm and the images with different color detection were integrated. The polarized MDCK cells grown in COM-free medium (to which 100 μg/ml COM crystals were added and incubated for 30 min, but were then removed) served as the control cells.