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. 2009 May 4;185(3):397–407. doi: 10.1083/jcb.200903088

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© 2009 Perpelescu et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 1. — RSF complex interacts with CENP-A chromatin at the mononucleosome level. (A) Ethidium bromide–stained gel showing DNA nucleosomal ladder of bulk chromatin that had undergone mild (200 U/ml × min, lane 1) and extensive (12,000 U/ml × min, lane 2) digestion with micrococcal nuclease in the presence of 0.3 M NaCl. Percentages of mono-, di-, and tri-nucleosomes for lane 2 are shown at the right. (B) The bulk chromatin of asynchronous HeLa cells was mildly digested with MNase as in A, immunoprecipitated using anti-CENP-A (lane 2), anti-CENP-H (lane 3), and anti-SNF2h antibodies (lane 4), and nonimmune IgG (lane 5). Input bulk chromatin, one-tenth of the other samples, was applied in lane 1. Samples were run on a 5–20% gel and immunostained with ACA serum on the same membrane. The apparent molecular weight of CENP-A is calculated as ∼17 kD. The bottom panel shows a Coomassie blue–stained duplicate SDS-PAGE gel with histones as loading controls. The ratio of CENP-A to histone H4 of lanes 2 and 4 relative to the input sample (lane 1) was calculated and is depicted at the bottom. (C) Identification of proteins recovered by nChIP with anti-SNF2h antibodies of bulk chromatin after mild digestion with MNase (A, lane 1). The proteins eluted using the antigen peptide p4c were separated on 7.5% SDS-PAGE, transferred to a PVDF membrane, and identified with Coomassie Brilliant blue staining (lane 1) or with anti-Rsf-1 (lane 2) and anti-SNF2h (lane 3) antibodies. Fig. S1 shows mass spectrometry analysis of an identical sample. The apparent molecular weight of Rsf-1 or SNF2h is calculated from the molecular weight marker as ∼250 kD or ∼135 kD, respectively. (D) The anti-CENP-A nChIP samples of mild (lane 1) and extensive (lane 2) MNase digestion were separated in a 5–20% gel and immunostained using ACA serum, and anti-Rsf-1 and -SNF2h antibodies. (E) Western blot analysis of the nChIP samples using antibodies against SNF2h (lanes 1 and 2), Rsf-1 (lanes 3 and 4), and control IgG (lanes 5 and 6) after mild (lanes 1, 3, and 5) and extensive (2, 4, and 6) MNase digestion of bulk chromatin.