FIG. 4.

In vitro nuclear import of an SV40-NLS-GFP fusion protein. HeLa cells grown on glass coverslips were permeabilized with digitonin and then incubated for 30 min with 2 μM of GFP fused to GST-SV40-NLS in the presence or absence of 2 μM kap α2, 2 μM kap β1, HeLa cytosolic extract, 500 μg/ml WGA, and an energy reconstitution system (500 μM GTP, 8 mM Ran, 2 μM ATP, 25 mM phosphocreatine, 30 U/ml creatine phosphokinase) as indicated. The permeabilized HeLa cells were fixed, and fusion proteins were visualized by direct fluorescence microscopy. Nuclei were stained with Hoechst 333258.