FIGURE 8:

AE1-4 and G^AE accumulate in the same compartment in GFP-Rab43–expressing cells. (A) COS7 cells were cotransfected with AE1-4, which has an extracellular V5 epitope tag, and GFP-Rab43 and subsequently infected with an adenoviral vector encoding G^AE-Ds. The cells were grown overnight at restrictive temperature and then shifted to permissive temperature for 30 min, at which time the cells were fixed, permeabilized, and stained with anti-V5 antibodies. After incubation with an Alexa Fluor 647–conjugated secondary antibody, the cells were imaged by confocal microscopy. The majority of AE1-4 accumulated in a compartment that was positive for both G^AE and GFP-Rab43. (B) COS7 cells were cotransfected with AE1-4 containing an extracellular V5 epitope tag and GFP-Rab43. At 24 h posttransfection, the cells were fixed, and nonpermeabilized cells were incubated with mouse anti-V5 antibodies to detect AE 1-4 on the cell surface. After extensive washing, the cells were permeabilized and incubated with rabbit antibodies specific for the cytoplasmic tail of AE1 and then with goat anti-mouse secondary antibody conjugated to Alexa Fluor 647 and goat anti-rabbit secondary antibody conjugated to Alexa Fluor 594. The cells were then imaged by confocal microscopy. AE1-4 accumulated on the surface of cells that did not express GFP-Rab43. However, in cells expressing GFP-Rab43 (indicated by arrows), AE1-4 was retained in an intracellular GFP-Rab43–containing compartment. (C) Quantification of the level of surface AE1-4 in Rab43-positive and negative cells. Values reflect the average value obtained from the analysis of at least 25 cells from two independent experiments. **p < 0.0001 (Student’s two-tailed t test). Scale bars, 5 μm.