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. 2014 Aug 15;28(16):1815–1826. doi: 10.1101/gad.243113.114

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© 2014 Deyter and Biggins; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 4. — CENP-A^Cse4 localizes to the euchromatin in psh1∆C cells. (A) Whole-cell extracts (WCE) from wild-type (WT) (SBY8851), psh1∆ (SBY8903), and psh1∆C-13Myc (SBY10921) cells expressing pGAL-3Flag-CSE4 were fractionated into soluble and chromatin fractions. Cse4 levels were monitored in each fraction with α-Flag antibodies. Pgk1 and dimethylation of histone H3 at Lys4 (H3K4Me₂) are markers of the soluble and chromatin fractions, respectively. (B) 3Flag-Cse4 was transiently overexpressed for 1 h in wild-type (SBY8851), psh1∆ (SBY8903), and psh1∆C-13Myc (SBY10921) cells, and its localization to chromatin was analyzed by immunofluorescence on chromosome spreads. DAPI staining was used to visualize DNA, while α-Flag staining revealed Cse4 localization. (C) Quantification of the percentage of chromatin masses in which Cse4 staining was coincident with total DNA staining instead of with kinetochores that contain discrete Cse4 foci. Wild type (SBY8851), n = 132; psh1∆ (SBY8903), n = 137; psh1∆C-13Myc (SBY10921), n = 130.