Figure 10. SETD3-dependent methylation of human β-actin that is overexpressed in S. cerevisiae.

Homogeneous recombinant rat SETD3 (rSETD3, 0.6 µg protein) or its human orthologue (hSETD3, 0.45 µg protein) were incubated at 37°C for 40 min in the presence of 1 µM (100 pmol, ≈220 × 10³ cpm) [¹H+³H]SAM and 2 µM (200 pmol, 8.9 µg) homogenous recombinant human β-actin produced in S. cerevisiae, and either purified in non-denaturing conditions as complexes with ATP (ATP-actin) or ADP (ADP-actin) or denatured and refolded to obtain soluble nucleotide-free protein (refolded nucleotide-free actin). Proteins were precipitated with 10% trichloroacetic acid to determine the incorporation of radioactivity. Values are the means ± range (error bars) of two independent measurements performed with two different β-actin preparations.