Fig. 4. TMD-dependent sorting of GFP TA proteins in polarised MDCK cells. (A-C) Filter-grown cells stably expressing GFP-17 were fixed and immunostained for PDI, with the use of Texas-Red-conjugated secondary antibodies. A single confocal section, visualised for GFP (A), PDI (B) or as a merge of GFP and Texas Red (PDI) fluorescence (C) is shown. (D-I) Filter-grown cells stably expressing GFP-22 were either fixed and immunostained for a 58 kDa basolateral protein of MDCK cells (Keller and Simons, 1998) with the use of Texas-Red-conjugated secondary antibodies (D-F) or incubated in the cold with biotinylated ConA administered in the upper compartment of a Transwell filter chamber, followed by Texas-Red-conjugated streptavidin and fixation (G-I). Panels (D-F) show a single confocal section (D, GFP alone; E, basolateral marker; F, merge of images in D and E). Note colocalization of the GFP-22 construct with the basolateral marker in this lateral plane. (G-I) x-z section, with GFP fluorescence shown in G, apical Con A staining in H and merged image I. Bar, 10 µm.

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