Coadministration of UO126 and UCN-01 in MM cells results in dephosphorylation and increased activation of p34^cdc2but not CDK2.

U266 and MM.1S cells were exposed to 20 μM UO126 ± 150 nM UCN-01 for 24 hours, after which cells were lysed, the proteins separated by SDS-PAGE, and total/phosphorylated p34^cdc2(p-cdc2) and CDK2 (cdk2) were monitored by Western blot analysis. For each condition, lanes were loaded with 30 μg of protein. Alternatively, kinase assays were performed after immunoprecipitation with p34^cdc2- and CDK2-specific antibodies as described in “Materials and methods.” The activity of p34^cdc2 and CDK2 was determined by monitoring incorporation of γ-[³²P] into histone H1, indicated by³²P-histone (IP: cdc2). The results of a representative experiment are shown; an additional study yielded equivalent results.