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Figure 2


Fig. 2. KAE1-Y359A is targeted to the plasma membrane in nonpolarised MDCKI cells and mistargeted to the apical membrane in polarised MDCKI cells. MDCKI cells stably expressing kAE1 or kAE1-Y359A were seeded on coverslips for nonpolarised cells (a,b) or polarised on filters (c-j). (a-c,g) kAE1 was detected using AE1 monoclonal antibody Bric170. (d,h) Rabbit anti-β-catenin antibody was used as a basolateral marker. The corresponding merged images for c and d, and g and h are shown in e and i, respectively. (f,j) An extracellular anti-AE1 antibody FITC-Bric6 was added to both the apical and basolateral surfaces of the intact cell monolayers. For the polarised images, the upper micrographs are focal planes taken parallel to the epithelium (X-Y) through the centre for kAE1 or the apical surface for kAE1-Y359A. The lower micrographs show focal planes perpendicular to the epithelium (X-Z) along the white line in the X-Y image. Normal kAE1 is at the plasma membrane in nonpolarised cells (a) and basolateral membrane in polarised cells using Bric170 (c) or FITC-Bric6 (f). The kAE1-Y359A protein is also at the plasma membrane in nonpolarised cells (b) but mistargeted to the apical membrane using Bric170 (g) or FITC-Bric6 (j). Scale bars: 30 µm.





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