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0343203325
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note
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2, 5 mM EGTA, 2 mM β-mercaptoethanol, 50 mM sucrose, and cycloheximide (50 mg/ml). Lysis, centrifugal clarification, and collection of the cytoplasmic fraction were carried out as described by Blow (33).
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38
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0343638939
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note
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Recombinant baculoviruses encoding His-tagged human cyclin B1s were gifts from D. O. Morgan. Purification was carried out as described by Kumagai and Dunphy (34). Each extract required titration with cyclin B to determine the appropriate concentration for observing effects on cell cycle timing.
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39
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0343203324
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note
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The Cdc25 antibody was a gift from E. Shibuya. The MPM-2 antibody was purchased from Upstate Biotechnology (Lake Placid, NY).
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40
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0343638940
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note
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We thank J. Yang for generous advice regarding these experiments; E. Evans and J. Yang for help with methodology; C. Mena and J. Simpson for technical support; S. Shenolikar, R. Abraham, E. Corcoran, S. Lemrow, and F. Winkler for their thoughtful comments on the manuscript; and J. Joseph and C. Kahl for provocative discussions during the course of the studies. Supported by NIH grant CA 82845 to A.R.M., by grants from Glaxo-Wellcome (ARM. and S.K.), and by a grant from the American Cancer Society to S.K. S.K. is a Scholar of the Leukemia Society of America and K.E.W. is a recipient of predoctoral fellowships from the NSF and the U.S. Department of Defense.
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