Cdk1-bound Cks proteins enhance APC/C phosphorylation and mitotic progression. (A) Cdk1–EYFP shows no distinct intracellular localization. M denotes metaphase alignment and onset of cyclin B1 destruction in U2OS cells. (B) Complementation of Cdk1 RNAi by RNAi-resistant Cdk1*–EYFP. (C) Reconstitution of Cdk1 depletion by Cdk1*–EYFP reveals cyclin B1 binding (Fig. S1 B and Video 1). Cdk1 localization gets dispersed upon cyclin B1 degradation at metaphase. (D) Complementation of Cdk1 depletion by non-Cks–binding Cdk1*–1N delays cells in mitosis. (E) Reconstituted expression of fluorescent wild-type Cdk1 rescues Cdk1 RNAi (Lindqvist et al., 2007); NEB to anaphase (NEB-A) is normal at ∼24 min (n = 8). Cdk1*–1N–EYFP-complemented cells are delayed in mitosis, NEB to anaphase is 57 min (n = 7). (right) Graph shows that Cdk1*–1N–EYFP-complemented cells delay independently of the spindle checkpoint. Knockdown of BubR1 (shBubR1; Lens et al., 2003) was verified by staining imaged cells with anti-BubR1 after fixation (not depicted). (F) Mitotic cells arrested in the spindle checkpoint were collected. Extracts were blotted with the indicated antibodies. (G) Intensities of the most phosphorylated forms of APC3 (ppp) and the unphosphorylated APC3 (u) from four different experiments as in F were quantified. A detailed description is shown in Fig. S1 E. WCE, whole cell extract. Error bars indicate standard deviations. Markers are given in kilodaltons. Bars: (A) 5 µM; (C and D) 10 µM.