An in vitro histone methyltransferase assay was performed as described previously (Shimazu et al., 2014 (link)). FLAG-tagged wild-type or mutant Suv39h1 was incubated in 1x Collin’s buffer (25 mM Tris-HCl pH 8.5, 5 mM DTT) with Histone H3 (0.5 μg) and 14C-labeled SAM (0.01 μCi, Perkin Elmer) for 15 min at 25°C. The reaction was stopped by adding Laemmli SDS sample buffer. Proteins were resolved on a 15% acrylamide SDS-PAGE gel, and the dry gel was exposed to an imaging plate (Fujifilm) for 1 day. Autoradiography was detected using a BAS-5000 Image Analyzer (Fujifilm).
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