Figure 9.

Cohesins can dissociate from chromatin in the absence of CDK1 activity. (A) Xenopus sperm nuclei (3,200 nuclei/μl) were incubated for 30 min at room temperature in interphase extracts from cycloheximide-treated Xenopus eggs before either 1 μM okadaic acid (left) or DMSO (right) was added. At different time points either extract samples (top) or chromatin reisolated from the reaction mixture by sucrose cushion centrifugation (bottom) were analyzed by SDS-PAGE and either PhosphorImaging (S³⁵CDC25) or immunoblotting with antibodies to the indicated proteins (all other panels). The cell cycle state of the extracts was analyzed by monitoring the behavior of ³⁵S-labeled CDC25 and the phosphorylation of histone H3 on serine 10 (H3P). (B) Phase contrast micrographs of human diploid fibroblasts grown to confluency and then treated for 2.5 h with 10 μg/ml cycloheximide and either with DMSO (right) or with 1 μM okadaic acid (left). Whole cell lysates (WCL) or chromatin pellets (CP) were then analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins (lower panels). Data from two different experiments are shown. OA, okadaic acid; H3P, histone H3 phosphorylated on serine 10.