Figure 5. An okadaic acid-sensitive phosphatase keeps Emi2 active to maintain CSF arrest.

(A) GST-Emi2 (FL) protein beads were incubated in CSF extracts to bind Cdc27. The beads were retrieved, washed, and then incubated in fresh buffer containing MgCl₂ and ATP in the presence of DMSO (−), okadaic acid (OA), recombinant Cdc2/Cyclin B1 or in combination as indicated. After incubation, the protein beads were pelleted, washed, and immunoblotted for Cdc27 and GST.

(B) Histone H1 protein was incubated with [γ-³²P] ATP and recombinant Cdc2/Cyclin B kinase in the presence or absence of or OA for 10 min. The reactions were separated by SDS-PAGE and the phosphorylation of Histone H1 was monitored by autoradiography.

(C) DMSO or 5 µM OA was added to CSF extracts. Samples were taken at the indicated times and the endogenous Cyclin B2 levels were examined by immunoblotting.

(D) CSF extracts were incubated with or without OA and the Cdc2 kinase activity of the extracts was examined using Histone H1 as a substrate. The reactions were separated by SDS-PAGE and the phosphorylation of Histone H1 was monitored by autoradiography.

(E) GST-Emi2 (FL) protein linked to glutathione Sepharose was incubated in CSF extracts supplemented with either DMSO or OA. After incubation, the protein beads were retrieved, thoroughly washed, and transferred into fresh CSF extracts supplemented with OA. Samples were taken at the indicated times to monitor Cyclin B2 levels by immunoblotting.

(F) ³⁵S-labeled Cyclin B1 and Emi2 proteins were synthesized in vitro and added to CSF extracts. After 15 min incubation, Ca²⁺, DMSO or OA (or both Ca²⁺ and OA) were added. Aliquots were removed at the indicated times and the degradation or gel mobility shifting of both Cyclin B1 and Emi2 were examined by autoradiography using a phosphorimager.