Fig. 3. Displacement of RII from Reh-RII but not Reh-RII(T54E) centrosomes after CDK1 phosphorylation. (A) Proteins from the Triton-X-100-insoluble fractions of Reh-RII and Reh-RII(T54E) interphase cells were incubated (for 45 minutes at 22°C in EBS phosphorylation buffer with 100 µM ATP) in the presence (lanes 3,5,7,9) or absence (lanes 2,4,6,8) of CDK1. After incubation, pellet (P; lanes 2-5) and supernatant (S; lanes 6-9; lower panel shows longer exposure) were separated by centrifugation and subjected to SDS-PAGE, and RII was immunodetected with anti-hRII mAb. The level of immunoreactive RII in each lane (in upper panel) was evaluated by densitometric scanning and relative intensities are given in the lower panel. As a positive control, 100 ng of human recombinant RII was incubated with CDK1 before the immunodetection (lane 1). (B) The localization of RII was analyzed by immunofluorescence in centrosome-nucleus complexes of interphase Reh-RII and Reh-RII(T54E) cells before and after incubation with CDK1. The location of RII and centrosomes was determined using anti-RII mAb (red) and anti-affinity purified anti-AKAP450 polyclonal antibodies (green). Inserts: DNA was stained with Hoechst 33342 (blue). Bars: 10 µm. (C) The percentage of centrosomes staining for RII before (-) and after (+) CDK1 incubation is given (n=200; * means P<0.001). One representative experiment of two or more is shown for all panels.