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Fig. 11. Polarized MDCK cells expressing V5-tagged AE1-4 or AE1-4L50A were fixed and stained with AE1-specific antibodies (A). Alternatively, polarized cells expressing V5-tagged AE1-4 were incubated at 4°C for 30 minutes in the absence (B-D) or presence (E-J) of 10 mM methyl-
-cyclodextrin. During the 4°C incubation, V5-specific monoclonal antibodies were added to the basolateral surface of these polarized cells. The cells were then washed and shifted to 37°C for 20 minutes in the absence (B-D) or presence (E-J) of 10 mM methyl--cyclodextrin/cholesterol. At this time, the cells were directly fixed (B-D and H-J) or they were shifted to citrate buffer, pH 1.5, for 5 minutes at 4°C to elute surface-bound antibodies prior to fixation (E-G). The cells were then permeabilized and incubated with a rabbit caveolin-1 antibody, followed by anti-mouse IgG conjugated to Alexa Fluor-594 and anti-rabbit IgG conjugated to Alexa Fluor-488. The xy-slice in each confocal image is approximately in the middle of the cell with the corresponding xz-slice below. The merged images in G and J indicate that AE1-4V5 polypeptides internalized as a result of cholesterol depletion/repletion accumulate in caveolin 1-positive endosomes. Black bars mark the position of the basal membrane. Bars, 10 µm.
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