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. 2008 Sep;19(9):3616–3624. doi: 10.1091/mbc.E08-01-0050

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© 2008 by The American Society for Cell Biology

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Figure 4. — Ubiquitylated H2B is associated with chromatin. Wild-type yeast cells (JR5-2A) were engineered by plasmid shuffle to express the indicated H2B-HA mutants (from a centromeric plasmid) as well as His-tagged G76A ubiquitin. Chromatin fractionation was then performed as described (Liang and Stillman, 1997). Whole cell extracts (WCE; lanes 1, 6, and 11) were subject to a low-speed centrifugation (Lo-SP) and separated into soluble material (SN; lanes 2, 7, and 12) and an insoluble pellet containing chromatin (PL; lanes 3, 8, and 13). The insoluble material was then treated with micrococcal nuclease (MN) to release mono- and polynucleosomes into the soluble fraction. These extracts were then subject to a second round of centrifugation to separate remaining insoluble material (PL; lanes 5, 10, and 15) from the solubilized nucleosomes (SN; lanes 4, 9, and 14). Nickel-affinity chromatography was then used to isolate ubiquitylated proteins in each fraction. H2B–HA in the Ni-NTA and input material, as well as Orc3 (control) in the input, were detected by WB.