A, in vitro kinase assays with purified CDK1/cyclin B complex and recombinant GST-PBK. RO3306 (5 μM) was used to inhibit CDK1 kinase activity. B, Conservation of PBK’s mitotic phosphorylation sites. C, in vitro kinase assays with purified CDK1/cyclin B complex to phosphorylate recombinant GST-PBK or GST-PBK-4A. D, In vitro kinase assays were done as in (A) except anti-phospho-PBK T9, T24, S32 and S59 antibodies were used.