Pcmv6 ac gfp plasmid

A plasmid encoding C-terminal green fluorescent protein (GFP)-tagged NDRG2 (pCMV6–AC–GFP-NDRG2) and a negative control pCMV6–AC-GFP plasmid without NDRG2 (mock plasmid) were purchased from OriGene (OriGene, USA). The competent Escherichia coli strains DH5α were used for proliferation of plasmid constructs. For each transformation, 100 ng of DNA was added to 25 μl of competent cells and incubated on ice for 30 min, followed by heat shock at 42 °C for 2 min and incubation on ice for 2 min. The cells were allowed to recover in 1 ml Luria-Bertani (LB) broth and then incubated for 60 min at 37 °C with shaking. Cells were plated on LB-agar plate containing 100 μg/ml ampicillin (plasmids encoded ampicillin resistance) and incubated at 37 °C overnight to select the transformants. After overnight culture, one colony of each plasmid was transferred to 3 ml of LB broth supplemented with ampicillin (50 μg/ml) for 5 hr of pre-culture at 37 °C before transfer to 500 ml LB broth for a further overnight of incubation in a rotating incubator. The overnight culture was centrifuged at 5000 g for 10 min, and the resulting pellet was used to extract plasmid DNA using PureLink™ HiPure plasmid filter Purification Kit (Invitrogen, UK) as per manufacturer’s instructions. The concentration of the DNA extracted was measured using the NanoDrop ND-100 spectrophotometer.

CHO cells were transiently transfected using Lipofectamine 2000 kit (Invitrogen), as per manufacturer’s protocol with the following ion channel coding vectors: hK_V1.1 (hKCNA1 gene) and hK_V1.2 (hKCNA2 gene) in pCMV6-AC-GFP plasmid (cat# RG211000 and RC222200; OriGene Technologies), hK_V1.5 in pEYFP plasmid (a kind gift from A. Felipe, University of Barcelona, Barcelona, Spain), hK_Ca3.1 (hKCNN4 gene) in pEGFP-C1 vector (a kind gift from H. Wulff, University of California, Davis, Davis, CA), hNa_V1.5 (hSCN5A, a kind gift from H. Abriel, University of Bern, Bern, Switzerland), and hHv1 (hVCN1, GenBank accession no. BC007277.1, a kind gift from Kenton Swartz, National Institutes of Health, Bethesda, MD). At 24 h after transfection, GFP-expressing transfectants were identified with Nikon TE 2000U fluorescence microscope using bandpass filters of 455–495 and 515–555 nm for excitation and emission, respectively, and used for current recordings (∼60–70% success rate for co-transfection). In general, currents were recorded 24–36 h after transfection.
Human embryonic kidney 293 cells stably expressing hK_V11.1 (hERG1 and hKCNH2 genes, a kind gift from H. Wulff), mK_Ca1.1 (BK_Ca, mKcnma1, a kind gift from C. Beeton, Baylor College of Medicine, Houston, TX), and hNa_V1.4 (hSCN4A gene, a kind gift from P. Lukács, Eötvös Loránd University, Budapest, Hungary) were used.