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. 2017 Jun 1;45(14):8314–8328. doi: 10.1093/nar/gkx503

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© The Author(s) 2017. 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-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 4. — Rap1 S731A mutation reduces its interaction with Rif1, but not Rif2. (A) Yeast two-hybrid assay indicated that rap1-S731A mutation significantly reduces, whereas rap1-S731D increases, the Rap1–Rif1 interaction. The Y axis shows the relative folds of β-galactosidase activity. Error bars indicate the s.d. (n = 4, *P-values < 0.05, **P-values < 0.001, Student's t-test, two-tailed). (B) Yeast two-hybrid assay indicated that WT and rap1-S mutants display similar Rap1-Rif2 interaction (n = 4, NS, non-significant, Student's t-test, two-tailed,). Bars, s.d. (C) Left panel, the aliquot of GST and GST-Rif1 (1709–1916) fusion proteins was resolved on SDS-PAGE and stained with Coomassie blue. Top-right panel, GST pulldown assay indicated that GST-Rif1 (1709–1916) significantly reduce its interaction with Rap1-S731A, whereas increase that with Rap1-S731D. Lower panel, the quantitative data of GST-Rif1 pulldown (n = 4, *P-values < 0.05, **P-values < 0.001, Student's t-test, two-tailed). Bars, s.d. (D) Left panel, the aliquot of GST and GST-Rif2 (1–395) fusion proteins was resolved on SDS-PAGE and stained with Coomassie blue. Top-right panel, GST pulldown assay demonstrated that the level of GST-Rif2 (1–395) interacting with Rap1 is not significantly different between WT and Rap1-S731A or Rap1-S731D, respectively. Lower panel, the quantitative data of GST-Rif2 pulldown (n = 4, NS, non-significant, Student's t-test, two-tailed). Bars, s.d.