Fig. 5. The tail-less construct EC0 is detected on the cell surface. (A) Schematic representation of HA-tagged wild-type E-cadherin (Ecad) and two mutant constructs: EC0, lacking the entire cytoplasmic domain; Ex, consisting of the extracellular domain but lacking the cytoplasmic and TM domains. (B) Confocal imaging of basolateral targeting of the tail-less EC0 construct. E-cadherin and endogenous Na⁺,K⁺-ATPase were detected by using DECMA-1and anti-Na⁺,K⁺-ATPase mAb. (C) Expression and basolateral targeting of EC0. (Upper panel) Immunoblot analysis of total cell lysates using DECMA-1 revealed that five times more EC0 was expressed than endogenous E-cadherin. (Lower panel) Cells expressing EC0 were grown on Transwell filters and their (a) apical or (b) basolateral membranes were biotinylated. After precipitating the biotinylated proteins using immobilized streptavidin, EC0 and endogenous E-cadherin were detected using DECMA-1. (D) No EC0 labeling was detected 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. Optical data were obtained as described in B. (E) EC0 does not form lateral dimers with endogenous E-cadherin. Cells expressing EC0 were lysed and incubated with either anti-HA or mAbs against the cytoplasmic domain of E-cadherin (C20820) preabsorbed with protein G-Sepharose. Proteins were separated by SDS-PAGE and detected using DECMA-1. (F) Immunofluorescence labeling of MDCK cells expressing EC81GFP with pan-cadherin antibodies. (G) The Ecad mutant construct Ex is released into both the apical and basolateral media. Cells expressing Ex were grown on Transwell filters. Secretion of Ex in the apical and basolateral media was assessed by immunoprecipitation with rabbit anti-E-cadherin antibodies and immunoblotting with DECMA-1. The barrier function of the transfectants was confirmed by the observation that mouse immunoglobulin was detected in the apical medium when added to the apical medium, but not when added to the basolateral medium (not shown). (H) Schematic representation of the full-length IL2 receptor chain construct (IL2R) and its derivatives. IL2RC: a construct lacking the entire cytoplasmic domain; IRCTME: a tail-less construct whose transmembrane domain was replaced with that of E-cadherin; IL2RECT: a construct whose cytoplasmic domain was replaced with that of E-cadherin; IL2RECTLA: a construct whose cytoplasmic domain was replaced with that of E-cadherin with the LA substitution. (I) Detection of IL2RC and IL2RCTME but not IL2RECT and IL2RECTLA on the apical membrane. Optical data were obtained as described in Fig. 5D except that anti-IL2R antibodies were used.