Figure 5.

Nuclear import of cyclin B1–Cdc2 is reduced by depletion of importin β. Nuclear import reactions were performed with cyclin B1ΔCRS–Cdc2K⁻ (A–C), wild-type cyclin B1–Cdc2 (D–F), or FITC-NLS-BSA (G–I) in the presence of mitotic cytosol (A, D, and G), mitotic cytosol that had been depleted of importin β (B, E, and H), or depleted cytosol supplemented with 0.5 μM importin β (C, F, and I). Cells were analyzed by direct immunofluorescence (G–I) or secondary immunofluorescence with monoclonal anti-cyclin B1 antibodies (A–F). (J) Western blot of importin β in the undepleted (lane 1) or depleted cytosol (lane 2). As shown (40, 41), addition of importin β to depleted cytosol did not restore import of NLS-BSA, presumably because other components (such as importin α) are also depleted with importin β.