FIG. 3.

Importin α in cytoplasmic extracts from primary macrophages promotes the nuclear import of Vpr. Monocytes were isolated from human PBMC. Macrophages were differentiated from monocytes by M-CSF. (A) Detection of importin α isoform mRNAs by real-time quantitative PCR. Total RNAs were extracted from monocytes and differentiated primary macrophages, and quantitative RT-PCR analysis was performed on a LightCycler system, using specific primers for each importin α isoform. Bars represent the mean values and standard errors for three experiments. (B) Detection by Western blotting of importin α proteins. Lysates containing 100 μg protein from monocytes and differentiated primary macrophages were subjected to Western blotting with MAbs against importins α1, α3, and α5 and against GAPDH as a control. The positions of the three importin α isoforms and GAPDH are indicated. (C) In vitro nuclear transport assay. Cytoplasmic extracts were prepared from monocytes and differentiated primary macrophages. Digitonin-permeabilized HeLa cells were incubated with 1 μM GST- and GFP-tagged N17C74 or GST- and GFP-tagged SV40 NLS and extracts containing 100 μg protein. After fixation, cells were analyzed by confocal laser scanning microscopy. Bar = 20 μm.