FIG. 4.

Importin α depletion prevents the nuclear import of Vpr in macrophages. (A) Lysates of differentiated primary macrophages were treated with a MAb against importin α1 or with preimmune normal mouse IgG, followed by treatment with protein A-Sepharose. The cytoplasmic extracts were clarified by centrifugation, and equivalent amounts of the immunodepleted extracts were subjected to Western blotting with MAbs against importins α1, α3, and α5, importin β, or GAPDH. The positions of the three importin α isoforms, importin β, and GAPDH are indicated. (B) In vitro nuclear transport assay. Digitonin-permeabilized HeLa cells were incubated with 1 μM GST- and GFP-tagged N17C74 or SV40 NLS and 100 μg of control or immunodepleted cytoplasmic extract. After fixation, cells were analyzed by confocal laser scanning microscopy. Bar = 20 μm.