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. 2008 Sep 22;28(23):7245–7258. doi: 10.1128/MCB.01085-08

FIG. 5.

FIG. 5.

ATM signaling is required for cyclin D1 phosphorylation following DSB induction. (A) Cyclin D1 degradation is attenuated in cells lacking ATM expression. Immortalized ATM+/+ or ATM−/− mouse embryonic fibroblasts (MEFs) expressing Flag-tagged cyclin D1 were gamma irradiated, and cyclin D1 levels were assessed. p-p53/total p53 induction served as markers of checkpoint activation in these cell lines. (B) DNA damage-induced cyclin D1 phosphorylation is only detected in cells expressing ATM. Immortalized ATM+/+ or ATM−/− MEFs expressing Flag-tagged cyclin D1 were irradiated as indicated, and lysates were precipitated with M2-agarose to detect Flag-tagged protein. Cyclin D1 phosphorylation and total levels were assessed by immunoblotting. (C) ATM signaling is required for cyclin D1 phosphorylation. NIH 3T3 cells were pretreated with DMSO or the specific ATM inhibitor KU-55933 for 30 min, followed by γIR and recovery for 15 or 30 min. Phosphorylated and total cyclin D1 were detected by immunoblotting. A second Western blot was performed with these lysates to probe for phosphorylated ATM and total ATM to ensure efficacy of ATM inhibition.

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