Fig. 4. Polarised expression of dominant dRTA kAE1 mutants. MDCKI-kAE1(R589H) cells (A), MDCKI-kAE1(S613F) cells (B), MDCKI-kAE1(R901Stop) cells (C,E) and MDCK1-kAE1 cells (D) were polarised on filters. (A-C) Cells were stained for AE1 using Bric 170 (green), (A-C, bottom panels) tight junctions were localised with anti-ZO-1 antibody (red). (D,E) FITC-Bric 6 antibody was added to apical and basolateral surfaces of the intact cell monolayer. (Top panels) Focal planes taken parallel to the epithelium near the centre of the cells (A,B,D) and at the apical surface (C,E). (Bottom panels) Focal planes perpendicular to the epithelium. (A-C, top) Staining with anti-AE1 Bric 170; (A-C, bottom) merged images of anti-AE1 (green) and anti-ZO-1 staining (red). Proteins kAE1(R589H) and kAE1(S613F) are both distributed throughout the polarised cells (A,B, respectively), whereas kAE1(R901Stop) is at the apical membrane (C) and was detected exclusively at the apical surface by FITC-Bric 6 (E). kAE1 was detected exclusively at the basolateral surface by FITC-Bric 6 on filters (D). Scale bar in C, 12 µm (for A-C); scale bar in E, 15 µm (for D,E); AP, apical; BL, basolateral.