HMTase activity of RE-IIBP. (A) Core histones were used as substrates in the HMTase assay with GST-RE-IIBP and GST-RE-IIBP point mutants (C483A and R477A). Methylation levels were quantified via filter binding assay, and data are represented as raw counts per minute incorporated. (B) Purified GST-RE-IIBP, GST-RE-IIBP point mutants, and GST proteins were incubated with HeLa cell extracts and subjected to HMTase assays. GST-bound beads were used as the negative control. The lower panel represents GST-RE-IIBP pulled down and immunoblotted with anti-G9a and anti-EZH2 antibodies. HeLa cell extracts were used as the positive control. (C) A similar HMTase assay as that described in panel A was performed with RE-IIBP deletion mutants (GST-RE-IIBP-1 and GST-RE-IIBP-2). A schematic representation of the domain structure of the recombinant RE-IIBP deletion mutants is shown. Conc, concentration; WT, wild type.