FIG. 5.
The αH1 and αH2 regions of Vpr play different roles in nuclear import. (A) Construction of plasmids that encoded GST- and GFP-tagged N17C74 and similarly tagged substitution mutants of N17C74. (B) Digitonin-permeabilized HeLa cells were incubated with GST- and GFP-tagged N17C74, αLAN17C74, and L67PN17C74. After fixation, cells were analyzed by confocal laser-scanning microscopy. Bar = 20 μm. (C) The lysates of HeLa cells that had been transfected with vectors that encoded Flag- and GFP-tagged full-length Vpr, αH1, αLA/αH1, αH2, and L67P/αH2, and Flag-tagged GFP, were incubated with glutathione-Sepharose beads coupled with GST-importin-α (lanes 2 to 5; 37.5, 75, 150, and 300 pmol, respectively) or GST (300 pmol), as indicated at the top of each panel. The proteins recovered on beads were separated by SDS-PAGE and detected by Western blotting with a Flag-specific MAb.