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In vitro methylation assay.In vitro methylation was performed by incubating GST-PRMTs and substrate (His)6-tagged proteins with Ado[methyl-3H]Met (77 Ci/mmol, Amersham) in a methylation buffer [50 mM potassium phosphate (pH 7.4), 0.4 mM EDTA and 200 mM NaCl] for 1 h at 30 °C. The methylation reaction was stopped by the addition of 5× SDS sample buffer. Following electrophoresis, gels were stained by Coomassie Brilliant Blue (CBB) R-250 for 30 min, destained in a 30% methanol (v/v)-10% acetic Z-DQMD-FMK (v/v) solution to visualize protein bands, and then soaked in isotope Amplify? solution (Amersham) for 30 min. Gels were dried by heat in a vacuum and radioactivity was visualized by fluorography (Hyper MP film, Amersham) at ?80 °C with an exposure time of 1 ～ 2 weeks.
Cell culture and transfection. Human embryonic kidney epithelial cells (HEK293T) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen, USA). One-hundred units/ml penicillin and 100 μg/ml streptomycin (Invitrogen, USA) were added as antibiotics. These cells were maintained at 37 °C under 5% CO2. DNA transfections were performed with Lipofectamine? reagents (Invitrogen, USA).