FIG. 5.

siRNA-induced down regulation of importin α isoforms prevents the nuclear import of Vpr. (A) Duplicate siRNA transfections corresponding to importins α1, α3, α5, and β, HPRT-specific siRNA transfection as a positive control, and nonspecific siRNA transfection as a negative control were carried out with HeLa cells. Cytoplasmic extracts were prepared from HeLa cells transfected with siRNA. The transfection efficiencies of siRNAs were determined by Western blotting using importin α1-, α3-, α5/7-, and β-specific antibodies. The positions of importins α1, α3, α5 (*), and β are indicated. (B) In vitro nuclear import assay. Digitonin-permeabilized HeLa cells were incubated with 1 μM GST- and GFP-tagged N17C74 or GST- and GFP-tagged SV40 NLS and 100 μg proteins from a cytoplasmic extract prepared from HeLa cells transfected with siRNA. After fixation, cells were analyzed by confocal laser scanning microscopy. Bar = 20 μm.