In vitro methylation of CF Im59 and CF Im68 subfragments. (A) The gels depicted on the middle and right-hand autoradiograms are exposed about 10-fold longer than the autoradiogram to the left. GST-68_GR_3-mut is GST-68_GR with all three arginines (202, 204, and 206) in the GAR motif mutated to alanines. (B) The [3H]SAM activity test with fraction 7 of Superdex-200 column. (C) The [3H]SAM activity test on the native and recombinant substrates. For an explanation of substrate names above the gel see Figure 1 and Supplemental Table 1. Assays were done in the presence of 1.3 pmol of H6-CF Im68, 1.2 pmol of native CF Im68, 4.4 pmol of H6-PABPN1, and 4 pmol of native PABPN1. Also included are 4.8 pmol of PRMT1 and PRMT5/WD45. The signal strength normalized to equal amounts of the substrate are indicated as a percent below the gel and the corresponding numbers are indicated by different fonts (normal, bold, and italic).
