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. 2015 Nov 17;44(1):315–329. doi: 10.1093/nar/gkv1260

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© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — Mcm10 binds Mcm2–7 complex in vitro. (A) 30 pmol of GST-Mcm10 or GST tag was incubated with increasing concentrations of radiolabeled PKA-Mcm2–7 at 30°C for 10 min in a GST pulldown assay. Results from similar experiments were quantified, averaged and plotted. (B) 30 pmol of GST-Mcm10 was incubated with increasing concentrations of radiolabeled PKA-Mcm2–7 in a GST pulldown assay. These experiments were performed with 5 mM ATP, 5 mM ATPγS or no nucleotide. Results from similar experiments were quantified, averaged and plotted. (C) 30 pmol of GST-Mcm10 was incubated with increasing concentrations of radiolabeled PKA-Mcm2, PKA-Mcm3, PKA-Mcm4, PKA-Mcm5, PKA-Mcm6 or PKA-Mcm7 at 30ºC for 10 min in a GST-pulldown assay. Results from similar experiments were quantified, averaged and plotted. (D) Glycerol gradient ultracentrifugation analysis of the Mcm10–Mcm2–7 complex. The fractions were analyzed and quantified by western blot using anti-His Tag antibody. Graphs from (A), (B) and (C) represent mean values from two independent experiments and error bars indicate the standard deviation of the mean.