Cyclin-dependent kinase 1-mediated phosphorylation of SET at serine 7 is essential for its oncogenic activity

a HeLa cells were treated with DMSO (control), taxol (100？nM for 16？h), or nocodazole (Noco, 100？ng/ml for 16？h). Total cell lysates were electrophoresed on regular and Phos-tag SDS polyacrylamide gels and probed with the indicated antibodies. Increased cyclin B1 levels marks cells in mitosis. An asterisk (*) marks the phosphorylated/shifted band. b HeLa cells were treated with nocodazole as indicated and cell lysates were further treated with (+) or without (？) λ phosphatase (ppase). Total cell lysates were probed with the indicated antibodies. Increased cyclin B1 levels marks cells in mitosis. An asterisk marks the phosphorylated/shifted band. c HeLa cells were treated with nocodazole, with or without various kinase inhibitors as indicated. Inhibitors were added 1.5？h before harvesting the cells (with MG132 to prevent cyclin B degradation and subsequent mitotic exit). The concentrations used for each inhibitor were as follows: VX680 2？μM, RO3306 5？μM, Purvalanol A 10？μM, BI-2536 100？nM, SB216763 10？μM, MK-2206 10？μM, SB203580 10？μM, U0126 20？μM, SP600125 20？μM, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 30？μM, and rapamycin 100？nM. Total cell lysates were electrophoresed on regular and Phos-tag SDS polyacrylamide gels and probed with the indicated antibodies. Increased cyclin B1 levels mark cells in mitosis. An asterisk marks the phosphorylated/shifted band. d In vitro kinase assays with purified CDK1/cyclin B1 complex using GST-tagged SET isoform 1 proteins as substrates. RO3306 (5？μM) was used to inhibit CDK1/cyclin B1 kinase activity. e GST-SET and GST-SET-S7A proteins were used for in vitro kinase assays with purified CDK1/cyclin B1 complex. f In vitro kinase assays were done as in e except anti-phospho-SET S7 antibody was use