Net 155h

1-beta-D-arabinofuranosylcytosine (AraC) and benzidine were obtained from Sigma-Aldrich. S-adenosyl-L-[methyl-³H] methionine (³H-AdoMet, 0.55 mCi/ml, NET-155H) and fluorographic enhancer, EN³HANCE, were from PerkinElmer. The pFlag-CMV2-p38α plasmid [3 (link)] was used as a template for PCR to generate mutations on R49 and R149. These plasmids were subsequently cloned into the pET6H vector for expression of recombinant proteins.

Methylation reactions were carried out in 20 μl methylation buffer (0.5 M EDTA, 1 M DTT and Tris-Hcl (8.0)) containing 0.5 uCi S-[³H-Me] adenosylmethionine (NET155H, Perkin Elmer), 1 μM recombinant PRMT9 protein and 3 μM MAVS or MAVS mutants. The reaction mixture was incubated at 30 °C for 5 min, 15 min, 30 min, 50 min, or 60 min then spotted onto Whatman 3 mm cellulose filter paper discs (Fisher Scientific). 5% ice-cold TCA was used to wash the paper discs for three times to stop the reaction, followed by 20% ethanol. Liquid scintillation counting was used to determine the amounts of radioactivity.

Whole-cell lysates from 3 × 10⁷ cells were prepared in F-buffer to immunoprecipitate MYC or ASH2L and the associated MTase. MYC- and ASH2L-associated MTase was measured with 5 μg core histones or GST-H3 N-terminal tails as substrates in the presence of 1.5 μl [³H]-SAM (Perkin-Elmer Life Sciences, NET-155H, 0.55 mCi/ml, 79.8 Ci/mmol) at 30°C for 30 min. Modified proteins were visualized by SDS-PAGE and autoradiogaphy. Site specificity and MYC-associated methyltransferase activity was measured with 1 μg recombinant histone H3 in the presence of 100 μM SAM at 30°C for 1 h. Histone H3 methylation was detected by immunoblotting with different H3K4 and H3K9 methyl-specific antibodies.