Pcdna3 myc dnmt1

Human DNMT1 and human DNMT3A cloned into pCAG-EGFP were kindly provided by Dr. Isao Suetake^{45 (link),46 (link)}. Human DNMT3B1 cloned into p3 × FLAG-CMV10 was kindly provided by Drs. Motoka Unoki and Hiroyuki Sasaki^{47 (link)}. Mutants of DNMT3B were generated by substituting serine for cysteine using the QuickChange Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer’s instructions using the following primers: C651S mutant 5′-GAT TGG CGG AAG CCC AAG CAA CGA TCT CTC AAA TG-3′ and 5′-CAT TTG AGA GAT CGT TGC TTG GGC TTC CGC CAA TC-3′, C716S mutant 5′-CAT CTC ACG GTT CCT GGA GAG TAA TCC AGT GAT TG-3′ and 5′-CAA TCA CTG GAT TAC TCT CCA GGA ACC GTG AGA TG-3′. A DNMT3B Q772A/F809A double mutant was generated by the megaprimer method using PrimerStar MAX Premix (Takara). F809A mutant 5′-CTC GAA AGG ATC GCT GGC TTT CCT GTG-3′ and 5′-GGG AGA TCT CTA TTC ACA TGC AAA G-3′, Q772A mutant 5′-CCC CTC GAG CTG CAG GAC TGC TTG GAA TAC AAT AGG ATA GCC AAG TTA AAG AAA GTA GAG ACA ATA ACC AAG-3′. All constructs were verified by sequencing. pcDNA3/Myc-DNMT1 (Addgene plasmid # 36939) and pcDNA3/Myc-DNMT3B1 (Addgene plasmid # 35522) were a gift from Arthur Riggs⁴⁸ . Each plasmid was transiently introduced into cells with polyethylene imine (PEI)-max (Polysciences) or Lipofectamine 2000 (Thermo Fisher Scientific), and the S-nitrosylation of proteins was analyzed after 24 h.