Characterization of WT and phosphomutant Cdt1 cell lines and proteins. (A) Thymidine synchronized and nocodazole arrested mitotic HeLa cells that were stably expressing HA-tagged Cdt1-WT or Cdt1-10D (Aurora B phosphomimetic mutant) were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies; IgG was taken as a negative control. 1% of the lysate was loaded as input. (B) Western blot from the cell lysate of Hela cells to analyze the expression levels of WT-Cdt1 protein along with indicated mutant versions. (C) SDS-PAG electrophoresis of purified GST-Cdt1-2E3D protein. (D) Asynchronous (left panel) or double thymidine synchronized HeLa cells stably expressing RNAi-resistant versions of the various Cdk1 (middle panel: WT, 5A, 2E3D) or Aurora B kinase (right panel: WT, 10A, 10D) mutants of Cdt1 were depleted of endogenous Cdt1 and DNA damage was assessed in each case after immunostaining using anti-γH2AX antibody (green) with the chromosomes counterstained using DAPI (blue). (E) Same as in D but in this case the interphase microtubule network was assessed only for the Cdk1 mutants (control, WT, 5A, 2E3D) after immunostaining the cells using an anti-tubulin antibody (red) with the chromosomes counterstained using DAPI (blue). Source data are available for this figure: SourceData FS4.