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. Author manuscript; available in PMC: 2024 Sep 17.
Published in final edited form as: J Immunol. 2009 Jul 20;183(4):2415–2424. doi: 10.4049/jimmunol.0804014

Figure 2. Nef traffics cell surface MHCII to lysosomal compartments.

Figure 2

A, U937 cells transfected to express either GFP alone or Nef and eGFP as indicated were surface labeled with anti-MHCII-biotin 12 h after transfection and then cultured for various times as indicated and subjected to the detection of either surface (open symbols) or total (closed symbols) label postpermeabilization. Mean fluorescence intensities were calculated for eGFP-gated cells and the data normalized to the starting intensities as a percentage of the residue of surface-labeled MHCII. The graphs show mean ± SD values of detected fluorescence label calculated from three independent experiments. B, U937 cells transfected to express eGFP (left column) or Nef-eGFP (right column) were surface-labeled 12 h later with anti-MHCII-biotin, and were either stained with labeled streptavidin-Alexa Fluor 568 immediately (0 h; top row) or cultured for 16 h before staining (16 h; bottom row), followed by confocal microscopic imaging. Scale bar, 10 μm. C, U937 cells transfected to express eGFP (left) or Nef-eGFP (right) were fixed 24 h after transfection and stained for MHCII (red; Alexa Fluor 568) and LAMP-1 (blue; Alexa Fluor 647), followed by confocal microscopy. Scale bar, 10 μm. D, U937 cells expressing Nef-eGFP were labeled for MHCII at the cell surface and cultured for various times as indicated. At each time point shown the surface label was stripped and cells were permeabilized and stained for the vesicular marker indicated. For detection of Arf6, U937 cells were cotransfected with plasmids expressing Nef-HA and Arf6-eGFP, and staining include hemagglutinin epitope detection. For tracking colocalization with dextran (Dex) or Tf, cells were given a 10-min pulse with fluorophores, Alexa Fluor 568-Tf or tetramethylrhodamine-Dex 3 h after surface labeling for MHCII. Cells were imaged by either high-resolution, wide-field fluorescence microscopy or confocal microscopy. Images of Nef-expressing cells were quantified for the fraction of internalized MHCII colocalizing with the various markers shown. Data are plotted as the extent of colocalization over time (mean ± SE; n = 100 cells).