Fig. 5. C-terminal domain of protein VI mediates import via importin α/β. (A) Western blot analysis of material bound to GST-p6c-NLS after GST pull-down from cytosol. Glutathione beads were saturated with either free GST (lane1 and 2) or GST-p6c-NLS (lanes 3 and 4) and incubated with cytosol in the presence of RanQ69L preloaded with GTP (lanes 1 and 3) or GDP (lanes 2 and 4). Bound material was examined by western blotting with antibodies against importin α (lower row) or importin β (upper row). (B) In vitro import of GST-p6c-NLS in permeabilized cells. Confocal images are shown. Detection of BSA-NLS-FITC was performed directly after fixation of cells (first column). To detect GST-p6c-NLS, indirect immunofluorescence with a specific antibody against the HA tag was carried out after fixation (second column). All reactions contained an energy regenerating system (except the reaction shown in b), and the recombinant transport factors Ran, NTF2, importin β and importin α (except the reaction shown in e, which lacks recombinant importin α). In b the reaction was depleted of energy by addition of hexokinase/glucose.