FIGURE 5.

PRMT7-catalyzed methylation of core histones with and without buffer change detected by fluorography. 4 μg of human recombinant histones H2A, H2B, H3.3, and H4, either in the supplied buffer or exchanged into the reaction buffer (see “Experimental Procedures”), or 4 μg of GST-GAR were mixed with PRMT7 (0.26 μm final concentration) and [³H]AdoMet and incubated at room temperature as described under “Experimental Procedures” for 20 h in a final volume of 40 μl. Samples were then mixed with SDS loading buffer, separated on 4–12% BisTris gel, and stained with Coomassie Blue (A). The protein substrate in each lane was labeled at the bottom of the graph. The expected position of molecular mass standards (Bio-Rad, broad range) is shown on the left in kDa. The fluoroimage was developed after 7 days (d) of film exposure (B) or 3 h of film exposure (C). As described under “Experimental Procedures,” the density of radioactive bands from the film shown in C (where the density was mostly in the linear range) was divided by the density of the Coomassie-stained polypeptide to obtain the specific radioactivity (D). Data were normalized against the specific radioactivity of H2B (with buffer change).