XB-IMG-136331
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FIGURE 6:. Deposphorylation of mitotic substrates in “collapsed” cells is a result of incomplete inhibition of Cdk-opposing phosphatases. (A) Cdk1/cyclin B1 activity does not drop in mitotic collapse cells. HeLa cells were synchronized at the S/G2 border and treated with the Wee1/Myt1 inhibitor, PD0166285, Cdc25 inhibitor, NSC663284, and the combination of the two in the presence of nocodazole. Cells were then collected at indicated time points and lysed. An aliquot of the lysate was analyzed by Western blotting for Nucleolin phosphorylation. β-Actin served as a loading control. Cyclin B1/Cdk1 complex was immunoprecipitated from the rest of the lysate and subjected to an in vitro kinase assay using histone H1 as a substrate. The kinase reaction mixture was resolved by SDS–PAGE, and the gel was exposed to phosphor-screen, which was then scanned with phosphor-imager. For a control, samples derived from the 4-h time point of DMSO-treated cells were treated with Cdk inhibitor (lane labeled “+Flavopiridol”), or processed omitting cyclin B1 antibody from immunoprecipitation (lane labeled “mock”). The gel was subsequently stained with Coomassie blue for loading. Panel on the right shows quantifications of histone H1 phosphorylation normalized to the 4 h time point of DMSO-treated cells. An average of three independent assays is shown. Error bars denote SD. (B) Simultaneous inhibition of Wee1/Myt1 and Cdc25 in cells already in mitosis does not cause mitotic substrate dephosphorylation. Mitotic HeLa cells were collected in nocodazole and then treated with Wee1/Myt1 and Cdc25 inhibitors for the indicated time, lysed, and analyzed by Western blotting. Mitotic substrates nucleolin and histone H3 remained phosphorylated throughout the experiment. (C) The phosphatase inhibitor, okadaic acid, prevents dephosphorylation of mitotic substrates in cells treated with a combination of Wee1/Myt1 and Cdc25 inhibitors. HeLa cells were synchronized at the S/G2 border after double thymidine block and treated with the Wee1/Myt1 inhibitor, PD0166285, and Cdc25 inhibitor, NSC663284, for the indicated time in the presence or absence of okadaic acid. Addition of the okadaic acid resulted in robust and sustained phosphorylation of mitotic substrates. Image published in: Potapova TA et al. (2011) © 2011 Potapova et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View |