The hypomethylated recombinant proteins Flag-KAP1 and Flag-ZNF224 so obtained were
incubated in presence of S-adenosyl-L-methyl-3H-methionine (3H-AdoMet). The incorporation of
radiolabeled methyl group on Flag-KAP1 and Flag-ZNF224 was evaluated by fluorography
(Figure 1c). As shown, a radioactive signal corresponding to the molecular weight of KAP1 was
detected in the presence of Flag-KAP1 and PRMT5 (lane 1), whereas no specific bands were observed
in the presence of Flag-ZNF224 and PRMT5 (lane 2), thus indicating that PRMT5 could specifically
methylate KAP1 in vitro. Arginine methylation of pooled histones was used as a positive control of the
in vitro methylation assay (Figure 1d).