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. 2005 Oct;16(10):5026–5039. doi: 10.1091/mbc.E05-05-0412

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Copyright © 2005, The American Society for Cell Biology

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Figure 7. — In vitro and in vivo phosphorylation of Mcm3 is dependent on the consensus CDK phosphorylation sites in the Mcm3 NLS3NES3 module. (A) GST-Mcm3 is phosphorylated by Cdc28-Clb2 kinase in vitro. In vitro kinase reactions were performed with purified Cdc28-Clb2 kinase mixed with purified GST-Mcm3, GST-Mcm3-cdk5A, or GST-Mcm3-cdk7A. Reaction products electrophoresed on SDS-PAGE were subjected to autoradiography (top) and Coomassie staining (bottom). (B) The CDK consensus sites in the Mcm3 NLS3NES3 are required for in vivo phosphorylation of Mcm3. YJL4110 (MCM3), YJL4313 (Myc₆-MCM3), YJL4324 (Myc₆-mcm3-cdk5A), and YJL4315 (Myc₆-mcm3-cdk7A) were metabolically labeled with ³²P-orthophosphate for 1 h before lysis and immunoprecipitation with 9E10 anti-Myc mAb. Immunoprecipitates were electrophoresed on SDS-PAGE and either subjected to autoradiography (top) or immunoblotted with rabbit anti-Myc polyclonal antibodies (bottom).