Arginine methylation in subunits of mammalian pre-mRNA cleavage factor I

In vitro activities of recombinant PRMT1 and the PRMT5/WD45 complex. Assays were done in the presence of 2 pmol each of substrates PABPN1, GST-68_GR, or D1/D2 and the reactions were incubated for 2.5 h with 0.32 μM PRMT1 (P1) or the 0.32 μM PRMT5/WD45 complex (P5W) as described in Materials and Methods. (A) For the [³H]SAM assay, 1.04 μCi (or 1.02 μM) [³H]SAM was complemented with an unlabeled SAM to a total concentration of 20 μM. Protein gels were blotted to nitrocellulose and exposed to PhosphorImager screens. (B,C) For Western blots, an unlabeled SAM (20 μM) was present in each reaction. The blots were probed with the sym10 and asym24 antibodies (1:2000 dilution).