Net155v250uc

The customized peptide arrays containing 214 ADMA-modified peptide sequences identified from LC-MS/MS were synthesized by Intavis Bioanalytical Instruments AG, Germany.^{25 (link)} Before the in vitro methylation reaction, H₂O was added to a 10 cm dish, and the peptide slides were placed on the top of the dish to allow them to hydrate in the hybridization oven for 3 h at 30 °C. Aliquots of 20 μg purified, recombinant PRMT1, 4, and 6 proteins and 40 μl radioactive S-adenosyl-L[methyl-³H] methionine (NET155V250UC, PerkinElmer) were mixed well in 200 μL methylation buffer (50mM Tris-HCl, pH 8.0, 100mM NaCl and 0.5mM EDTA) and incubated on the slides for 2 h at 30 ºC. The slides were washed three times with PBS (5 min/each time) and then treated with amplify (NAMP100V, GE Healthcare, Waukesha, WI) while gently shaking at room temperature for 45 min. Subsequently, the slides were dried before exposing to an X-ray film in the dark at −80ºC. The methylation of the peptide array was detected using autoradiography. A negative control well, devoid of any peptide sequence, was utilized as a reference for quantification.

Methyltransferase activity assays for G9a and GLP were performed by monitoring the incorporation of tritium-labeled methyl group to lysine 9 of H3 (1–25) peptide using scintillation proximity assay (SPA). The enzymatic reactions were performed at 23 °C with 20 min incubation of 10 μL of reaction mixture in 25 mM potassium phosphate pH 8.0, 1 mM EDTA, 2 mM MgCl₂, 0.01% Triton X-100 containing 8 μM of cold SAM, and 2 μM of ³H-SAM (catalogue no. NET155 V250UC; PerkinElmer; www.perkinelmer.com), 1 μM of biotinylated H3 (1–25), 5 nM G9a or GLP, and compound titrations from 1.5 nM to 25 μM. To stop the reactions, 10 μL of 7.5 M guanidine hydrochloride was added, followed by 60 μL of buffer (20 mM Tris, pH 8.0), mixed, and transferred to a 384-well streptavidin coated Flash-plate (PerkinElmer, http://www.perkinelmer.ca). After mixing, the mixtures in Flash-plate were incubated for 2 h, and the CPM counts were measured using Topcount plate reader (PerkinElmer, www.perkinelmer.com). The CPM counts in the absence of compound for each data set were defined as 100% activity. In the absence of the enzyme, the CPM counts in each data set were defined as background (0%). All enzymatic reactions were performed in triplicate, and IC₅₀ values were determined by fitting the data to Four Parameter Logistic equation using GraphPad Prism 7 software.