Figure 7. In vitro β-actin methylation in the presence of purified recombinant rat or human SETD3 overexpressed in E. coli.

Mammalian SETD3 proteins were produced in E. coli and purified by affinity chromatography on nickel-Sepharose (HisTrap HP), as described in the 'Materials and methods' section. Recombinant rat SETD3 (rSETD3, 0.4 µg protein) or its human orthologue (hSETD3, 0.4 µg protein) were incubated at 37°C for the indicated times in the presence of 1 µM (100 pmol, ≈230 × 10³ cpm) [¹H+³H]SAM and 2 µM (200 pmol, 8.9 µg) homogenous recombinant human β-actin or its mutated form (H73A). Proteins were precipitated with 10% trichloroacetic acid to determine the incorporation of radioactivity. The figure shows the results of single experiments.

Figure 7—figure supplement 1. (A) SDS-PAGE and (B) Western-blot analysis of fractions obtained during the purification of recombinant human SETD3 produced in E. coli.

Human SETD3 was purified by affinity chromatography on nickel-Sepharose (HisTrap HP) as described in the 'Materials and methods' section. For the SDS-PAGE analysis, 15 µl of sample from each fraction was loaded onto a 10% gel, electrophoresed and the resulting gel was then stained with colloidal Coomassie blue. For the Western-blot analysis, 3 µl of each fraction was loaded onto a 10% gel, electrophoresed and blotted to nitrocellulose membrane, which was then sequentially probed with a mouse primary antibody against His₆ tag and a horseradish peroxidase-conjugated goat anti-mouse antibody. The secondary antibody was detected by employing enhanced chemiluminescence. M, prestained protein marker; L, cell-free lysate of E. coli applied on the column; FT, flow through; W, wash. Fractions 60 to 300 were eluted with the indicated concentrations of imidazole.