Fig. 7. NgCAM tail 45-59 is sufficient for sorting to the apical membrane in MDCK cells. (A) Diagrammatic depiction of LDLR-based NgCAM cytoplasmic tail chimeras. Chimeras contain the proximal (amino acid residues 5-27) basolateral sorting signal of LDLR that is co-linear with the endocytosis motif and part of the NgCAM cytoplasmic tail as indicated. (B) Fully polarized MDCK cells were transiently transfected with cDNA encoding NgCAM and incubated at 37°C for 30 hours. Cells expressing NgCAM were surface stained with anti-NgCAM antibody (8D9). Subsequently, cells were fixed, permeabilized and incubated with secondary antibodies labeled with Alexa 594. The locations of the apical, basal and lateral domains are indicated by arrows for easier orientation. (C-E) Polarized MDCK cells were transiently transfected with cDNAs encoding Lct27 (C), Lct27Nct45-59 (D) and Lct27Nct66-114 (E), and incubated at 37°C for 30 hours. The transfected constructs were visualized by surface staining with anti-LDLR antibodies (C7). After surface staining, cells were fixed, permeabilized and stained for an endogenous basolateral marker protein using the anti-gp58 antibody. Cells were then incubated with secondary antibodies labeled with Alexa 488 (gp58) and Alexa 594 (Lct27 constructs). Specimens were analyzed by confocal microscopy, and representative x-z sections are shown.