823534.fig.001a
(a)
823534.fig.001b
(b)
823534.fig.001c
(c)
823534.fig.001d
(d)
Figure 1: H3K79me3T80ph antibody specificity. (a) Shown are the three H3 isoforms and their amino acid sequence in the core of the protein surrounding lysine 79 and threonine 80 (emphasized) (top), and the biotinylated peptides designed for this study that are either unmodified, contain mono-, di-, or trimethylation of K79 plus phosphorylated T80, or phosphorylated T80 alone (bottom). (b) Histones from HeLa cells were isolated and subjected to western blotting with the H3K79me3T80ph antibody alone (no peptide) or in the presence of 15 µM of biotinylated H3 (71–88) peptides with no modification, K79 trimethylation and T80 phosphorylation, T80 phosphorylation alone, or an H3 (1–21) S10ph peptide. (c) 50, 100, 150, 200, 250 nmoles of biotinylated H3 (71–88) peptides containing mono-, di-, or trimethylated K79 in conjunction with T80 phosphorylation were spotted onto nitrocellulose and subjected to H3K79me3T80ph and Avidin-HRP western blotting. (d) 50, 100, 150, 200, 250 nmoles of biotinylated H3 (71–88) peptides with K79 trimethylation and T80 phosphorylation or mono-, di-, or trimethylated K79 alone were spotted onto nitrocellulose and subjected to H3K79me3T80ph and avidin-HRP western blotting.