Pcdna3 plasmid vector

Mouse Inpp4a cDNAs with C-terminal FLAG tag were generated by PCR using mouse cerebellum cDNAs (wild-type, Inpp4a^ΔEx1,2 KO and Inpp4a^ΔEx23 KO mice) as templates. The primers were 5′-GGGGTACCCCCCACGTGGTCCAAAAGCAAG-3′ (sense), 5′-ATAAGAATGCGGCCGCAAGCTTTCACTTGTCATCGTCATCCTTGTAGTCTGTCTCAACTTTTCCGTAAGTCCCT-3′ (antisense for Inpp4a WT, Δ2, Δ23) and 5′-ATAAGAATGCGGCCGCAAGCTTTCACTTGTCATCGTCATCCTTGTAGTCCGGGCACTTTTGTCTGCCTC-3′ (antisense for Inpp4a CB). TaKaRa LA Taq (TaKaRa Bio.) was used for the PCR reactions. The PCR products containing the full-length Inpp4a cDNAs with FLAG tag were cut with Asp718 (Roche) and NotI (Nippon Gene) and then subcloned into pcDNA3 plasmid vector (Invitrogen). The produced plasmids are referred to as pcDNA3-mouse Inpp4a-FLAG, pcDNA3-mouse Inpp4a ΔEx1,2-FLAG, pcDNA3-mouse Inpp4a ΔEx23, and pcDNA3-mouse Inpp4a CB-FLAG plasmids. Sequencing was performed on both strands.

The N406K mutation was generated using QuickChange site-directed mutagenesis kits according to the manufacturer’s instructions (Stratagene, La Jolla, CA) and was engineered into the common splice variant of human cardiac voltage-dependent Na channel SCN5A/hNav1.5 [containing a glutamine at position 1077, we note as Q1077 (Genbank accession no. AC1377587)] in the pcDNA3 plasmid vector (Invitrogen, Carlsbad, CA) as previously reported [11,12]. All clones were sequenced to confirm integrity and to ensure the presence of the target mutations without other substitutions.