Figure 2.
SETD3 is active on Met73. A, in vitro methylation reactions with SETD3, indicated full-length actin proteins, and radiolabeled methyl donor ([3H]SAM). The reactions were resolved by SDS–PAGE (bottom panel), and activity was visualized by autoradiography (top panel). B, overnight reaction of SETD3 activity on actin peptides containing His73, Lys73, or Met73, by two assays: Promega MTase-GloTM assay measures the reaction by-product (SAH) concentration (y axis) and autoradiography (3H peptides). C, SETD3 activity on Met73 as a function of pH (red points for below pH 9 and blue points for above pH 9). D, SETD3 activity on Met73 is linear as a function of time. E, single-turnover kinetics of SETD3 on Met73 under pH 8 for 1-h reaction. F, ITC measurements of SETD3 against Met73 and His73 (for original graphs, see Fig. S1, B and C). G, inhibition of SETD3 activity on His73 methylation by Met73-containing peptide.