Figure 4.
The phosphorylation of Cdt1 by the Cdk1 kinase interferes with Ska1-binding. (A) Cdk1 phosphorylation sites on Cdt1 that were mutated to obtain phophomemeitic and non-phosphorylatable versions of the same protein. (B) Western blot showing co-immunoprecipitation of HA-Cdt1 constructs and the Ska1 complex (detected by the SKA1 antibody). (C) Quantitative band intensities from B. Data presented as mean ± SD. (D) WT (Di), 2E3D mutant (Dii), or in vitro Cdk1-phosphorylated Cdt1 (Diii) were run on a Superose 6 gel filtration column after mixing with equimolar ratio of the Ska1 complex for 1 h, as described in Materials and methods. Recorded elution profiles of the above-mentioned mixture of proteins/complexes exiting the column, as indicated. The elution profiles of Cdt1 variants or the Ska1 complex run individually have not been shown. (E) Western blots of fractions from Di, Dii, or Diii above probed using primary antibodies as indicated on the right, for the proteins/protein complexes run separately or in combination as indicated on the left. The initial two fractions (21 and 22) of the elution for Cdt1-WT + Ska1 complex have been marked with a red box to highlight the observed difference with those of the individually run proteins/complexes or a mix of Cdt1-2E3D + Ska1 complex (marked in blue). (F) Same as E but with the phosphomimetic mutant variant of Cdt1 or the Ska1 complex as indicated. (G) BLI sensograms showing wavelength shifts (nm) generated by the addition of 0.5 μg/ml Cdt1-2E3D protein with increasing concentrations of SKA1 (0.3–10 µM), as indicated in the plot. (H) BLI sensograms of the indicated Cdt1 variant binding to SKA1 used at 0.6 µM concentration. (I) Nocodazole arrested mitotic HeLa cells that were stably expressing HA-tagged Cdt1-WT or Cdt1-2E3D (Cdk1 phosphomimetic mutant) were immunoprecipitated (IP) with the antibodies indicated on the right. Source data are available for this figure: SourceData F4.

The phosphorylation of Cdt1 by the Cdk1 kinase interferes with Ska1-binding. (A) Cdk1 phosphorylation sites on Cdt1 that were mutated to obtain phophomemeitic and non-phosphorylatable versions of the same protein. (B) Western blot showing co-immunoprecipitation of HA-Cdt1 constructs and the Ska1 complex (detected by the SKA1 antibody). (C) Quantitative band intensities from B. Data presented as mean ± SD. (D) WT (Di), 2E3D mutant (Dii), or in vitro Cdk1-phosphorylated Cdt1 (Diii) were run on a Superose 6 gel filtration column after mixing with equimolar ratio of the Ska1 complex for 1 h, as described in Materials and methods. Recorded elution profiles of the above-mentioned mixture of proteins/complexes exiting the column, as indicated. The elution profiles of Cdt1 variants or the Ska1 complex run individually have not been shown. (E) Western blots of fractions from Di, Dii, or Diii above probed using primary antibodies as indicated on the right, for the proteins/protein complexes run separately or in combination as indicated on the left. The initial two fractions (21 and 22) of the elution for Cdt1-WT + Ska1 complex have been marked with a red box to highlight the observed difference with those of the individually run proteins/complexes or a mix of Cdt1-2E3D + Ska1 complex (marked in blue). (F) Same as E but with the phosphomimetic mutant variant of Cdt1 or the Ska1 complex as indicated. (G) BLI sensograms showing wavelength shifts (nm) generated by the addition of 0.5 μg/ml Cdt1-2E3D protein with increasing concentrations of SKA1 (0.3–10 µM), as indicated in the plot. (H) BLI sensograms of the indicated Cdt1 variant binding to SKA1 used at 0.6 µM concentration. (I) Nocodazole arrested mitotic HeLa cells that were stably expressing HA-tagged Cdt1-WT or Cdt1-2E3D (Cdk1 phosphomimetic mutant) were immunoprecipitated (IP) with the antibodies indicated on the right. Source data are available for this figure: SourceData F4.

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