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. Author manuscript; available in PMC: 2009 Dec 8.
Published in final edited form as: Curr Biol. 2007 Feb 6;17(3):213–224. doi: 10.1016/j.cub.2006.12.045

Figure 6. Emi2 associates with PP2A acting in opposition of Cdc2/Cyclin B kinase activity.

Figure 6

(A) DMSO, 1 or 3 µM OA was added to CSF extracts. Aliquots were removed at the indicated times and immunoblotted for endogenous Cyclin B2.

(B) GST or GST-Emi2 (FL) protein beads were incubated in CSF extracts. After incubation, the protein beads were retrieved, washed, and immunoblotted for PP2A (top) and PP1 (bottom).

(C) IgG or PP2A antibody was immobilized on Protein A Sepharose beads and incubated in CSF extracts. The beads were retrieved, washed and mixed with GST-Emi2 (aa 489–651) WT protein that was pre-phosphorylated with recombinant Cdc2/Cyclin B kinase in the presence of [γ-32P] ATP. The phosphorylation of GST-Emi2 (aa 489–651) WT was examined by autoradiography after 0, 30 and 60 min.

(D) OA was added into CSF extracts supplemented with either GST, GST-Emi2 WT, or GST-Emi2 T545/551A proteins. At the indicated times, samples were withdrawn and immunoblotted for endogenous Cyclin B2.

(E) GST-Emi2 protein beads (aa 489–651; WT or T545/551A) were incubated in CSF extracts in the presence of DMSO (−) or OA. After incubation, the protein beads were retrieved, washed, and immunoblotted for Cdc27 and GST.