Figure 6. In vitro β-actin methylation in the presence of purified recombinant rat or human SETD3 overexpressed in COS-7 cells.

Homogeneous recombinant rat SETD3 (rSETD3, 0.4 µg protein) or its human orthologue (hSETD3, 0.3 µg protein) were incubated at 37°C for the indicated times in the presence of 1 µM (100 pmol, ≈300–400 × 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 results of representative experiments from two independent experiments performed.

Figure 6—figure supplement 1. (A) SDS-PAGE and (B) Western-blot analysis of fractions obtained during the purification of the recombinant rat SETD3 protein produced in COS-7 cells.

Rat SETD3 protein was purified to homogeneity 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 and electrophoresed, and the resulting gel was then stained with silver (Shevchenko et al., 1996). For the Western-blot analysis, 7.5 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 (27-4710-01, GE) and a horseradish peroxidase-conjugated goat anti-mouse antibody (A2554, Sigma-Aldrich). The secondary antibody was detected by employing enhanced chemiluminescence. M, prestained protein marker; L, cell-free lysate of COS-7 cells applied on the column; FT, flow through; W, wash. Fractions 60 to 300 were eluted with the indicated concentrations of imidazole.