Fig. S4

Type I protein arginine methyltransferase activity of zebrafish Prmt8. (A) GST-fused recombinant zebrafish Prmt8 was expressed in Escherichia coli and purified. In vitro methylation was conducted with Prmt8 and recombinant mouse fibrillarin as the methyl-accepting protein in the presence of 1.5 μCi of [methyl-³H]-AdoMet (60 Ci/mmol, Amersham Biotech) at 37°C for 60 min in a total volume of 15 μl in reaction buffer (50 mM sodium phosphate, pH 7.5). The samples were subjected to SDS-PAGE. The gels were then stained, treated with EN³HANCE (Perkin Elmer) and dried for fluorography. Control reactions with methyl-accepting protein (fibrillarin) or methyltransferase (GST-ZF8) only were conducted. (B) Reduced asymmetric dimethylarginine polypeptide signals in zebrafish prmt8 morphants. Embryos injected with 2 ng of MO2 (MO) or not (WT) were collected at 48 hpf. Fifty microgram of embryonic extract protein was subjected to western blot analyses with an asymmetric dimethylarginine-specific antibody (ASYM24). β-actin was used as a loading control.