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. 2010 Dec 31;286(10):8361–8368. doi: 10.1074/jbc.M110.204115

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© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 2. — HKMT assay of NSD1-CD. A, nucleosomes assembled with recombinant, unmethylated wild-type or mutant histones (H3K18A, H3K27A, H3K36A, and H4K20A) or nucleosomes bearing methyl analogs mimicking different degrees of methylation on H3K36 (H3K36Me1, H3K36Me2 and H3K36Me3). Top panel, ³H fluorography using [³H]AdoMet as the methyl donor. Bottom panel, Coomassie Brilliant Blue (CBB) staining of histones. The concentration of NSD1-CD used for the assay was 0.1 μm, and that of nucleosomes in the reactions was 0.35 μm. B, HKMT activity on recombinant histone octamers and nucleosomes. Increasing concentrations (0.03, 0.1, and 0.3 μm) of NSD1-CD were titrated in the assay. Top panel, ³H fluorography shows that histones H3, H2A/2B, and H4 were methylated using histone octamers, whereas only H3K36 was methylated when nucleosomes were used as substrates. Middle panel, Coomassie Brilliant Blue staining of histones. Bottom panel, Coomassie Brilliant Blue staining of NSD1-CD.