14c labeled sam

One microgram of FLAG-tagged EF1A1 were incubated in 1× Collin's buffer (25 mM Tris-HCl pH 8.5, 5 mM DTT) with His-METTL10 (1 µg) and 14C-labeled SAM (0.01 µCi, Perkin Elmer) for 2 h to overnight at 30°C. The reaction was stopped by adding Laemmli SDS-sample buffer. Proteins were resolved on a 12.5% acrylamide SDS-PAGE gel, and the dry gel was exposed to an imaging plate (FUJI-FILM) for 48 h, and the autoradiography was detected using a BAS-5000 Image analyzer (FUJI-FILM).

FLAG-tagged RPL3 was transiently expressed in METTL18 KO cells and immunopurified with ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich). The purified proteins were incubated in 1× reaction buffer (50 mM Tris-HCl pH 8.5 and 50 mM MgCl₂) with 1 µg of His-GST-METTL18, 2 µM MTAN, and 0.01 µCi of ¹⁴C-labeled SAM (PerkinElmer) at 30°C for 2 hr. The reaction was stopped by the addition of Laemmli SDS-sample buffer. Proteins were separated on a 10% acrylamide SDS-PAGE gel. The dried gel was exposed to an imaging plate (FUJIFILM) for 48 hr. The autoradiograph was detected with a phosphor imaging scanner (Cytiva, Amersham Typhoon).

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).