Figure 5. Rab7 is required for lysosomal delivery of MHCII removed from the surface in Nef-expressing cells.

A, U937 cells were either singly transfected or cotransfected for expression of hemagglutinin epitope (HAp) alone or F2-Nef-HAp or WT Rab7 or DN Rab7 in combinations as indicated. Cells were stained with anti-MHCII-biotin 12 h later and cultured for various times as shown before the detection of residual cell surface label. Mean fluorescence intensities were calculated for HAp-gated cells and the data normalized to the starting intensities as a percentage of the residue of surface-labeled MHCII. B, U937 cells transfected to express Nef-GFP with either WT Rab7 or DN Rab7-eGFP (blue) were fixed 24 h after transfection and stained for MHCII (green; Alexa Fluor 647) and LAMP-1 (red; Alexa Fluor 568) followed by confocal microscopy. Insets show gray-scale images of the same cells for cyan and blue colors as identified by the inset frames. C, Nef-HAp-expressing cells from the experiment shown in B above were quantified for the fraction of internalized MHCII colocalizing with LAMP-1 in the presence of WT Rab7 or DN Rab7 (mean + SE; n = 50 cells).