Fig. 6. The N-terminal repeats of the E-cadherin extracellular domain are required for the basolateral localization. (A) Schematic representation of EC0 and its derivatives each carrying a deletion of one of the five extracellular domains. (B) Expression of the EC0 derivatives. Constructs were detected using antibodies as indicated. (C) Basolateral localization of the EC0 derivatives. Cells were grown on Transwell filters, and either the (a) apical or (b) basolateral membranes were biotinylated. After the biotinylated proteins were precipitated using immobilized streptavidin, the constructs were detected using DECMA-1 except for EC0EC4, which was detected with ECCD-2. Constructs lacking either EC1, EC2 or EC3 were detected on the apical membrane. (D) Detection of EC1, EC2 and EC3, but not EC4 and EC5 on the apical membrane. Cells grown on Transwell filters were fixed and incubated with DECMA-1 without permeabilization to detect E-cadherin on the cell surface. To detect the EC0 deletion constructs EC0EC4 and EC0EC5, ECCD-2 was used because DECMA-1 cannot recognize these constructs on the cell surface. Constructs EC0EC1, EC0EC2 and EC0EC3 were detected on the apical membrane. Optical data were obtained as described in Fig. 5D. (E) Quantitative analysis of the apical membrane targeting. Relative amounts of the pool of E-cadherin at the apical membrane (not shown) and of its derivatives as described in Fig. 5D, Fig. 6D,G, and Fig. 8D were quantified using NIH Image and expressed as a percentage of the total cell surface pool of the protein. In addition to EC0 derivatives lacking EC1, EC2 or EC3, another EC0 construct containing a W2A substitution (EC0WA) was detected on the apical membrane. (F) Immunoblot detection of EC0WA and EWA. Total cell lysates were analyzed by using DECMA-1. (G). Detection of EC0WA but not EWA on the apical membrane. Optical data were obtained as in Fig. 5D.