Amplify namp100v

For peptide methylation experiments, the H3K36 (27-43: Ac-APATGGVKKPHRYRP-NH2) and the ssK36 (27-43: Ac-AGKTGGVKRPNNYRS-NH2) peptides were purchased from Royobiotech (Shanghai, China) in HPLC grade purified form with >95% purity. 6 μM NSD2 were used for the methylation of 300 µM H3K36 peptide substrate and 10 times less NSD2 enzyme for the methylation of the ssK36 peptide used in a concentration ranging from 1.2 to 300 μM in 20 μl methylation buffer (50 mM Tris/HCl pH 8.5, 50 mM NaCl and 0.5 mM DTT) supplemented with 0.76 μM radioactive labeled AdoMet (Perkin Elmer). Reactions were conducted overnight at 25 °C. The reactions were stopped by the addition of 2X Tricine-SDS-PAGE loading buffer and incubation for 5 min at 95 °C. Afterward, the samples were separated by Tricine-SDS-PAGE, which was followed by the incubation of the gel in amplify NAMP100V (GE Healthcare) for 1 h on a shaker and drying of the gel for 2 h at 70 °C in vacuum. The signals of the transferred radioactive labeled methyl groups were detected by autoradiography using a Hyperfilm TM high performance autoradiography film (GE Healthcare) at -80 °C in the dark. Film development was performed with an Optimus TR developing machine. Band intensities were quantified by ImageJ 58 and background corrected.

The methylation of non-histone substrate proteins and recombinant H3.1 (NEB) was performed in methylation buffer (50 mM Tris/HCl pH 8.5, 50 mM NaCl and 0.5 mM DTT) supplemented with NSD2 and 0.76 µM labeled [methyl-3 H]-AdoMet (Perkin Elmer Inc., dissolved at 25 µM in 10 mM sulfuric acid) for 4 h at 25 °C. For methylation of the H3K36 (29-43)-GST and ssK36 (29-43)-GST proteins, 420 nM NSD2 was added and the mixture incubated overnight at 25 °C. The methylation reaction was stopped by the addition of SDS loading buffer and boiling for 5 min at 95 °C. NSD2 and target protein concentrations in the individual experiments are indicated in the figure legends. Equal amounts of target protein and K-to-R mutant protein was confirmed by Coomassie Brilliant Blue staining and Western Blot using as primary antibody anti-GST (GE Healthcare, 27457701 V). The methylated samples were separated by 12% SDS-PAGE. Then, the SDS gel was incubated for 1 h in Amplify NAMP100V (Ge Healthcare) and dried for 90 min at 65 °C under vacuum. The dried SDS gel was exposed to Hyperfilm TM high performance autoradiography films (GE Healthcare) in the dark at -80 °C. Film development was performed with an Optimus TR developing machine. Band intensities were quantified by ImageJ 58 and background corrected.

For the preparation of the peptide arrays the SPOT synthesis method and an Autospot peptide array synthesizer (Intavis AG, Köln, Germany) were used. 24; 35 The arrays contained fifteen amino acid long peptides spotted on a cellulose membrane. The membrane was pre-incubated in methylation buffer (50 mM Tris/HCl pH 9, 2.5 mM MgCl2, 4 mM DTT) for 5 minutes and then incubated in methylation buffer supplemented with 0.76 µM radioactively labeled AdoMet (Perkin Elmer) and different concentations of RomA (7.5-60 nM) for 60 minutes. This step was followed by 5 times washing for 5 minutes with wash buffer (100 mM NaHCO3, 1% SDS) and incubation for 5 minutes in amplify NAMP100V (GE Healthcare). Then, the membrane was exposed to a Hyperfilm TM high performance autoradiography film (GE Healthcare) at -80°C in the dark for different periods of time. The film was developed using an Optimax Typ TR machine.