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. 1999 Aug 17;96(17):9622–9627. doi: 10.1073/pnas.96.17.9622

Figure 2.

Figure 2

Importin α is translocated into the nucleus in the presence of CAS and Ran-GTP. Nuclear transport of importin α was assayed in digitonin-permeabilized cells for 30 min at 22°C in the presence of recombinant transport factors. A preformed importin α⋅β complex was mixed with either Ran-GDP (A) or RanQ69L-GTP (B) in the presence of an energy-regeneration system before addition to the cells. Similarly, importin α was mixed with buffer alone (C), RanQ69L-GTP alone (D), CAS alone (E), or CAS and RanQ69L-GTP (F). Before addition to the cells, mixes were incubated on ice for 3 min to allow for complex formation. ATP, GTP, and an energy-regeneration system were not added in reactions C–F. Cells were fixed immediately with paraformaldehyde, and importin α was detected by secondary immunofluorescence (35). The following final concentrations of factors were used in these assays: 1 μM importin α, 1 μM importin β, 2 μM CAS, and 2.5 μM Ran. Nuclei were scanned with a confocal fluorescence microscope (63× oil objective).