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. Author manuscript; available in PMC: 2011 Jul 28.
Published in final edited form as: Oncogene. 2010 Jan 11;29(11):1702–1716. doi: 10.1038/onc.2009.464

Figure 6.

Figure 6

SP600125 suppresses the activation of Cdk1-cyclin B1 kinase in G2 cells. (a) SP600125 suppresses de-phosphorylation of Cdk1 at Tyr15 but does not affect Cdk1 protein levels. HCT116 cells released from thymidine synchrony were treated with 20 μM SP600125 (SP) at 4 h after release from thymidine block. Nocodazole (NOC) was added at 1 h after release. Cell extracts prepared before release (Thy) and at the indicated times after release from thymidine synchrony were examined for cyclin B1, Cdk1, and phosphorylation of Cdk1 at Tyr15 by immunoblotting with specific antibodies. (b) SP600125-dependent suppression of Cdk1-associated kinase activity. Cells released from thymidine synchrony were treated with nocodazole and SP600125. Cdk1-associated kinase activity was measured in Cdk1 immunoprecipitates of cell extracts prepared at the indicated times after release from thymidine synchrony. Histone H1 (H1) was used as a substrate. (c) SP600125-dependent suppression of cyclin B1-associated kinase activity. Cells released from thymidine synchrony were treated with nocodazole and SP600125. Cyclin B-associated kinase activity was measured in cyclin B immunoprecipitates from cell extracts prepared at different times. Cyclin B-associated kinase activity increases in control cells coincident with cells entering mitosis. Histone H1 was used as a substrate. (d) SP600125 suppresses the activation of Aurora A and Polo-like kinase 1 in G2 cells. Cells released from thymidine synchrony were treated with nocodazole and SP600125 as in (a). Aurora A-associated kinase activity was measured in Aurora A immunoprecipitates of cell extracts using myelin basic protein (MBP) as a substrate. Plk1-associated kinase activity was measured in Plk1 immunoprecipitates of cell extracts using α-casein as a substrate. Western blot analysis of cell extracts prepared before release (Thy) and at the indicated times after release from thymidine synchrony with Aurora A- and Plk1-specific antibodies is shown.