Pcmv6 an gfp

The plasmid-expressing 3xFlag-tagged human Pur-α cDNA (NCBI accession number: NM_008989) was generated by subcloning Pur-α cDNA into the vector pCMV6-AN-3DDK (OriGene). The plasmid encoding Myc-tagged mouse DUSP8 was generated by cloning the Dusp8 cDNA into the vector pMTSM-Myc (6 (link)). The plasmid expressing DUSP8 (C246S) phosphatase–dead mutant was generated from the Myc-DUSP8 plasmid by mutating cysteine-246 to serine using PCR-based site-directed mutagenesis. The plasmids expressing YFP-fused DUSP8 and CFP-fused Pur-α proteins were constructed by subcloning the Dusp8 cDNA and Pur-α cDNA from the Myc-Dusp8 plasmid and 3xFlag-Pur-α plasmid into the pCMV6-AC-Yfp vector (OriGene) and pCMV6-AN-Cfp vector (OriGene), respectively. The plasmid expressing GFP-fused Pur-α protein was generated by subcloning the human Pur-α cDNA into the vector pCMV6-AN-Gfp (OriGene). The plasmids encoding Pur-α (S217A) and Pur-α (T183A) mutant proteins were generated by mutating the indicated amino acid residues in the 3xFlag-Pur-α or GFP-Pur-α plasmid.

Wild-type human Cav3.1b cDNA was donated by T. Snutch (Vancouver, BC, Canada) (GenBank accession number AF134986.1), HA-Cav3.1 and Kir2.1 cDNA from E. Bourinet (Institut de Génomique Fonctionnelle, Montpellier, FR), and CaM cDNAs by J. Adelman (Vollum Institute, OR, USA). GFP-Cav3.1 and GFP-αCaMKII were subcloned into a GFP mammalian vector (pCMV6-AN-GFP) (OriGene Technologies, Rockville, MD, USA) and mKate-CaM prepared from a pmKate2-N mammalian expression vector (Axxora, Farmingdale, NY, USA). CaMKIIN was amplified from a mouse hippocampal cDNA library (RIKEN) and cloned into the pcDNA3.1 vector (Addgene, Cambridge, MA, USA) [24 (link)]. To create a deletion of the C-terminus, PCR with specific primers was performed on Cav3.1 outside of the C-terminus region, followed by sequencing and subcloning into the pCMV6-AN-GFP vector (OriGene Technologies, Rockville, MD, USA) or the pcDNA3.1 vector (Addgene, Cambridge, MA, USA) and sequenced for final confirmation. The GFP and mKate tags were attached to the N-terminal regions of target proteins. To create the Cav3.1 pore mutant, a single point mutation PCR was performed on E354K in the pore region with the primer sequences: 5′-ACAGGTCGTTGAGCCGC-3′, followed by sequencing and subcloning into the pcDNA3.1 vector (Addgene, Cambridge, MA, USA) and sequenced for final confirmation.