Prolonged mitotic duration in importin-β 45–462-transfected cells requires Mad2 activity. (A) WB analysis of Mad2 protein in cells collected by shake-off after cotransfection with importin-β 45–462 and either pSUPER vector (lane −), or pSUPER-Mad2, expressing Mad2-specific shRNAs (lane +). (B) The induction of mitotic delay in video-recorded cells transfected with importin-β 1–876 or 45–462 requires Mad2. (C) Examples of: (a) A vector-transfected cell, completing mitosis in ∼1.5 h; and of cells cotransfected with either importin-β 45−462 and pSUPER vector (b, note chromosome oscillations and prolonged duration); or with importin-β 45−462 and pSUPER-Mad2 (c), exiting mitosis in less than 1 h with abnormal chromosome segregation. Images were acquired as described for Fig. 3. Bars, 10 µm.