Figure 3: Anti-EED (aa429–441) antibody disrupts PRC2 and attenuates its histone methyltransferase activity.
From: The central role of EED in the orchestration of polycomb group complexes
(a) Anti-EED (aa429–441) antibody inhibits the EED–EZH2 interaction. Halo-tagged full-length EED protein was incubated with EZH2 in the presence of different amounts of control IgG, anti-EED (aa429–441) and anti-EED (aa186–304) antibodies. Halo-Link PD (pull down) followed by immunoblot analysis is shown. (b) Anti-EED (aa429–441) antibody inhibits PRC2-mediated methylation of histone H3 in vitro. Indicated amounts of purified PRC2, control IgG, anti-EED (aa429–441) and anti-EED (aa186–304) antibodies were incubated with recombinant histone H3 with unmethylated H3K27 for 2 h. Immunoblot analyses with anti-H3K27me3 and anti-H3K27me2 antibodies were then performed. (c) Anti-EED (aa429–441) antibody inhibits PRC2-mediated methylation of nucleosomes in vitro. Indicated amounts of purified PRC2, control IgG, anti-EED (aa429–441) and anti-EED (aa186–304) antibodies were incubated with nucleosomes containing unmethylated H3K27. ELISA with anti-H3K27me3 antibody was then performed to measure PRC2 histone methyltransferase activity. Assays were repeated three times in triplicate. All error bars represent ±s.e.m.
