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. 2013 Jun 15;24(12):2034–2044. doi: 10.1091/mbc.E12-12-0893

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© 2013 Boeckmann et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — Centromeric association of phosphorylated Cse4. (A) Specificity of Cse4 phospho antibody (αp-Cse4). Western blots of Cse4 proteins purified from strains (BY4741) expressing wild-type Cse4 (pMB1601), cse4-4SA (pMB1566), or cse4-4SD (pMB1563) were probed with αHA (loading control) or αp-Cse4. (B) Phosphatase treatment of Cse4 reduces affinity of αp-Cse4 to Cse4. Cse4 protein purified from BY4741 expressing wild-type Cse4 (pMB1601) was treated with or without CIP and analyzed by Western blots probed with αHA (loading control) or αp-Cse4. (C) Phosphorylated Cse4 is associated with centromeres. ChIP of extracts treated with (CIP) or without CIP (no CIP) was performed with αHA or αp-Cse4 (YMB8378) and assayed by PCR for CEN3 and CEN4 sequences. The graph shows an example of a single experiment. Analysis of four independent experiments and statistical significance are shown in D. (D) Ratios of αHA/input DNA and αp-Cse4/αHA signals normalized to the no-CIP condition. Four independent biological replicates were performed. Error bars show the mean ± 95% confidence intervals. Statistical significance was determined by paired t test. Open symbols, CEN3; filled symbols, CEN4. (E) Centromere-associated phosphorylated Cse4 is increased in nocodazole-treated cells. ChIP samples from wild-type strain (YMB8378) treated with α-factor or nocodazole were analyzed as described in C. The αp-Cse4 signals (normalized to αHA) before and after CIP treatment were calculated. Results from two independent experiments were pooled. Lines on the scatter plots show the mean ± SE of the mean. Statistical significance was determined by one-way analysis of variance (ANOVA). Open symbols, CEN3; filled symbols, CEN4. (F) Phosphorylation of Cse4 is increased in cells depleted of Scc1. ChIP was performed on strain YMB8674 arrested in metaphase (Cdc20 OFF) with defective cohesion (Scc1 OFF). The control strain (Scc1 ON) was grown in galactose medium. Fraction of phospho-Cse4 is the fraction of the total αp-Cse4 signal removed by CIP treatment. Results from two independent experiments were pooled and plotted as in D. Significance was determined by t test. Open symbols, CEN3; filled symbols, CEN4.