Schematic representation of the nuclear import assay. (A) The classic assay. Cells are permeabilized with digitonin, which leaves the nuclear envelope intact. The cytosol is washed out and nuclear import is reconstituted by addition of the protein of interest (labelled fluorescently), cytosolic extracts or nuclear import receptors, the Ran system (RanGDP, NTF2, RanGAP, RanBP1) and an energy regenerating system. Accumulation of the protein of interest is examined by confocal microscopy. (B) Adapted assay. The assay is carried out as described above but purified and labelled HIV-1 RTCs are used instead of the protein of interest.