Figure 1.

Mitosis-dependent nuclear accumulation of cyclin B1–Cdc2 requires phosphorylation sites in the CRS. Digitonin-permeabilized HeLa cells were incubated for 30 min with preformed complexes of Myc-epitope-tagged cyclin B1 and kinase-deficient Cdc2K⁻ (300 nM), plus either interphase cytosol (A, C, E, and G) or mitotic cytosol (B, D, F, and H). Complexes contained either wild-type cyclin B1 (A and B), a deletion mutant lacking the CRS domain (B1ΔCRS) (C and D), a mutant in which four Ser residues in the CRS are changed to Glu (B1E) (E and F), or a mutant in which these residues are changed to Ala (B1A) (G and H). Cells were fixed and analyzed by secondary immunofluorescence with antibodies against the Myc epitope tag.