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. 2023 Nov 1;51(22):12185–12206. doi: 10.1093/nar/gkad934

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© The Author(s) 2023. 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 (https://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 6. — PCNA post-translational modification in the context of MSH2 MSH3 overexpression occurs at the K164 residue. MSH2 and MSH3 or MSH2 and empty vector were overexpressed following galactose induction in a His-POL30, His-pol30K164R, His-pol30K242R, or His-pol30K164R + K242R, background. Aliquots were collected and fixed for flow cytometry analysis before (0h) and after (17h) induction. After induction, cells were harvested for TCA extraction and anti-PCNA western blot. (A) Histograms are shown of chromosomal content of asynchronous populations before and after induction with galactose. Flow cytometry experiments were repeated at least three times, with at least two independent transformants. 1C indicates 1× DNA content; 2C indicates 2× DNA content. (B) Quantification of relative proportion of cells in different phases of the cell cycle. The percentage of cells in G1 (1C), G2/M (2C) or S (between 1C and 2C) phases was determined using BD FlowJo software (see Supplementary Figure S2 for details). Plotted values correspond to data collected from at least three independent experiments. Error bars represent SEM. Pairwise t-tests comparing the number of cells in G1 and S phase following MSH2 MSH3 O/E in His-POL30 versus His-pol30 alleles were performed. The counts were significantly different for His-POL30 versus His-pol30K164R (P< 0.0001 for G1 and S phase cells) or His-pol30K164R + K242R (GI: P = 0.0098, S: P< 0.0001). In contrast, the cells counts were not significantly different between His-POL30 and His-pol30K242R (G1: P = 0.8497; S: P = 0.8314). (C) PCNA western Blot. Note that the PCNA and M-PCNA of his-POL30 strains migrates at a higher weight due to the His-tag. These data are from a single blot, but not adjacent lanes, as indicated by the vertical lines. (D) PCNA western Blot Quantification as described above. Pairwise t-tests indicated significant difference in the proportion of M-PCNA in His-POL30 compared to His-pol30K164R or His-pol30K164R + K242R (p<0.0001) but not compared to His-pol30-K242R (P = 0.4152).