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. 2015 May 15;29(10):1058–1073. doi: 10.1101/gad.259432.115

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© 2015 Fang et al.; 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 2. — CENP-N specifically binds to open CENP-A chromatin but not to compact CENP-A chromatin (see also Supplemental Fig. S3). (A) In vitro mononucleosome-binding assay. Mononucleosomes (0.15 µM) were incubated with His-CENP-N^1–289mono in different amounts and separated by 6% native PAGE gel. (B) In vitro polynucleosome-binding assay. Polynucleosomes (0.15 µM) were incubated with His-CENP-N^1–289 in different amounts and analyzed by 1.0% agarose gel. (C) Statistical analysis of the proportion of nucleosome binding as shown in B. (D) In vitro polynucleosome-binding assay. Polynucleosomes (0.15 µM) were incubated with His-CENP-C^426–943 in different amounts and analyzed by 1.0% agarose gel. (E) Statistical analysis for the proportion of nucleosome binding as shown in D. (F) In vitro mononucleosome-binding assay. Mononucleosomes (0.15 µM) were incubated with His-CENP-N^1–289 in different amounts in the absence or presence of MgCl₂ and analyzed by 6% native PAGE gel. (G) In vitro polynucleosome-binding and sucrose gradient assay. Chromatin was incubated with His-CENP-N^1–289 in a molar ratio of 1:2 and analyzed by sucrose gradient sedimentation. Each sample was fractionated by sucrose gradient centrifugation, and DNA collected from individual fractions was sized on agarose gel.