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Figure 6


Fig. 6. Pervanadate-induced tyrosine phosphorylation influences basolateral kAE1 localisation. MDCKI-cells stably expressing kAE1 (A,B,C,D) or four apically targeted kAE1 mutants kAE1Y359A (E), Nt{Delta}1-360AE1 (F), kAE1(Y904A,Y907A) (H) or kAE1R901Stop (G) polarised on filters. 200 µM pervanadate (PV) was added to the cells in B-C, E-H or 514 mM NaHCO3 added to D for the times indicated. kAE1 was detected using AE1 monoclonal antibody Bric170 and a suitable secondary antibody. The upper micrographs are focal planes taken parallel to the epithelium (X-Y) near the centre for kAE1 or at the apical membrane for the kAE1 mutants. 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 basolateral membrane in untreated control cells (A) and those treated with pervanadate for 15 minutes (B). kAE1 localisation was intracellular after 30 minutes of pervanadate treatment in C. (D) NaHCO3 had no effect on kAE1 localisation. (E-H) Pervanadate treatment for 30 minutes did not alter the apical localisation of kAE1 mutants that lack either Y359 or Y904. Scale bar: 30 µm.





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