Fig. 2: Mina53 catalyzes demethylation of H4R3me2a in vitro and in cells.

a Representative dot-blotting analysis of in vitro demethylation reactions in the presence of wildtype (WT) Mina53 or the catalytically inactive mutant Mina53 and synthetic peptides. b Representative dot-blotting analysis of in vitro demethylation reactions in the presence or absence of various reaction components. Representative mass spectra of demethylation products in reactions with H4R3me2a (c) or H4R3me1 (d) as substrates. Reactions were performed in the presence of WT or the catalytically inactive mutant Mina53 in parallel. Representative immunoblotting analysis of H4R3 methylation status and PRMT1 in HEK293T cells stably expressing WT or the catalytically inactive mutant Mina53 (e), or stably depleting endogenous Mina53 (f). g A representative conformation of the N terminus of H4-Mina53 complex in the H4R3me2a form, obtained from the MD simulation. The electrostatic surface of Mina53 is illustrated, with negatively charged areas in red and positively charged areas in blue. The H4R3 peptide is shown as spheres: R3me2a is colored green, the positively charged residues (K5, K8, K12, K16, and R17) are colored blue, and the other residues are colored white. The experiments related to (e, f) have been performed in duplicate independently.