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. 2004 Jul 8;23(15):3113–3121. doi: 10.1038/sj.emboj.7600308

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Copyright © 2004, European Molecular Biology Organization

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Analysis of individual phosphorylation site mutants. (A) In vitro phosphorylation of various recombinant Bub1 proteins by MAPK. Lanes 1–6, Coomassie staining of the substrate proteins; lanes 7–12, autoradiogram of the in vitro phosphorylation reaction. WT, wild-type Bub1. (B) Immunoblot of various Bub1 proteins associated with chromosomes. Mitotic chromosomes were isolated from extracts containing wild-type or various Bub1 mutants as indicated on the top. Extracts (lanes 1–8) or chromosomal fractions (lanes 9–16) were immunoblotted with anti-Bub1 antibody. (C) Spindle checkpoint function of various Bub1 proteins. Extracts containing various Bub1 proteins as indicated were incubated with 10 000 nuclei/μl and 1 ng/μl nocodazole. Samples were taken for histone H1 kinase measurement at times indicated at the bottom. An autoradiogram of the reaction is shown.