Contribution of Cdk1–Cks1 to Clb5 early degradation. (A) Binding of Cdk1 by Clb5-GFP, Clb5-2D-GFP (I166D and F291D), and Clb5-3D-GFP (F169D, F254D, and F291D). Lysates of cells expressing wild-type or mutant Clb5-GFP from the endogenous CLB5 promoter were incubated with GFP-binding protein coupled to Sepharose beads. After washing, associated proteins were analyzed by SDS-PAGE and Western blotting with anti-GFP and anti-Cdk1 antibodies simultaneously. (B) Degradation profiles of wild-type Clb5-GFP, Clb5-3D-GFP, and Clb5-3D-GFP-Cks1, as in Fig. 3 A; n > 70 cells per strain. (C) Degradation profiles of securin-2A-Cks1-GFP, securin-2A-GFP, and Clb5-GFP in the securin-2A-Cks1 or securin-2A background, as in Fig. 3 A; n > 50 cells in the Clb5-GFP strains and n > 90 cells in the securin-GFP strains. In the inset, n > 100 cells per strain. (D) Fraction of securin-2A or securin-2A-Cks1 remaining when spindle elongation occurs, as in Fig. 4 B; n > 100 cells per strain.