Fig. 6. The surface distribution and steady-state localization of wild-type and mutant F_c38-63 constructs in polarized MDCK cells. Polarized MDCK cells stably expressing F_c38-63 (A,B) or F_c38-63Y47A (C) or transiently expressing F_c38-63Y47L (D) or F_c38-63L50A (E) were grown on Transwell filters. The intact cells were either incubated with the F_c-receptor-specific antibody for 1 hour at 4°C, washed and fixed in 3% paraformaldehyde (A) or the cells were fixed in 3% paraformaldehyde, permeabilized by incubation in PBST and incubated with the F_c-receptor-specific antibody (B-E). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine (A-E) and phalloidin conjugated to FITC (B-E). Following washing, the distribution of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The 0.5 µm xy image in each panel is near the center of the cells. Regions that are yellow in B-E indicate significant overlap in the distribution of the chimera and actin. The black arrowhead next to each panel marks the position of the basal membrane in the xz image. Bars, 10 µm.