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Pcmv6neo

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The PCMV6NEO is a plasmid vector that contains the neomycin resistance gene under the control of the cytomegalovirus (CMV) promoter. It can be used for the selection of mammalian cells transfected with the vector.

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Lab products found in correlation

Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions) X tremegene 9, by Roche (1 mentions) Horseradish peroxidase hrp conjugated anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions) Pgfp 5 rs nt scr, by OriGene (1 mentions) Quikchange lightning multi site directed mutagenesis kit, by Agilent Technologies (1 mentions) Hurat1, by OriGene (1 mentions)

4 protocols using pcmv6neo

1

Overexpression Assay of EPAC1 and EPAC2

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We obtained MYC tagged-EPAC1 from Dr. Stephen J Yarwood of Heriot-Watt University, Edinburgh, UK and DDK tagged-EPAC2 (#RC205335) from Origene (Rockville, MD). 2.5x105 cells/ml in triplicate wells in 6-well plates were transfected with 5μg empty vector plasmid (#PCMV6NEO, Origene, Rockville, MD) or MYC tagged-EPAC1 or DDK tagged-EPAC2 plasmids using Lipofectamine™ RNAiMAX (Thermo Fisher Scientific, Waltham, MA) in reduced serum Opti-MEM. The medium was changed 16 hours post transfection, and the cells were maintained in Geneticin (G418 2.5mg/ml) containing medium. Cells were lysed 5 days post- transfection and protein expression was analyzed by western blot using anti-Myc and ant-DDK antibodies. Bulk transfected cells were trypsinized and plated in 6 replicate wells (5,000 cells/well) in multiple 96-well plates and the cell growth was assessed using MTT assay.
Krishnan A., Bhasker A.I., Singh M.K., Rodriguez C.I., Castro-Pérez E., Altameemi S., Lares M., Khan H., Ndiaye M., Ahmad N., Schieke S.M, & Setaluri V. (2022). EPAC Regulates Melanoma Growth by Stimulating mTORC1 Signaling and Loss of EPAC Signaling Dependence Correlates with Melanoma Progression. Molecular cancer research : MCR, 20(10), 1548-1560.
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2

Overexpression of AEG-1 in Cell Lines

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Cells were seeded in 6-well culture plates at a density of 1.5 × 105 per well in 2 ml complete culture medium. After 24 h, the cells were transfected with 1.5 μg of either pCMV6-Neo (Origene, Rockville, MD, USA) or with pCMV6-Neo MTDH (Origene, NM_178812, CW101031) using X-tremeGENE 9 (Roche Diagnostic Corporation, Indianapolis, IN, USA) according to the manufacturer's instructions. After 24 h, fresh medium was added to the cells or the cells were seeded for follow-up experiments. Upregulation of AEG-1 was confirmed by qPCR (Supplementary Materials and Methods) and western blot (Supplementary Figure 2).
Gnosa S., Zhang H., Brodin V.P., Carstensen J., Adell G, & Sun X.F. (2014). AEG-1 expression is an independent prognostic factor in rectal cancer patients with preoperative radiotherapy: a study in a Swedish clinical trial. British Journal of Cancer, 111(1), 166-173.
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3

Cyclin D1 and ETV4 Regulation Protocol

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Antibody against Cyclin D1 (rabbit polyclonal) was purchased from Cell Signaling Technology (Beverly, MA). Anti -ETV4 (rabbit polyclonal) antibody and respective anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies were procured from Santa Cruz Biotechnology (Santa Cruz, CA). The β-actin (mouse monoclonal) antibody was purchased from Sigma-Aldrich (St. Louis MO). ETV4 knockdown short hairpin RNA (shRNA) expression constructs (pGFP-V-RS-shETV4) along with scrambled control (pGFP-V-RS-NTScr) and ETV4 overexpression construct (pCMV6-ETV4) and scramble control (pCMV6-Neo) were purchased from Origene (Rockville, MD). Cyclin D1 overexpression construct (pCMV6-CCND1) and control vector (pCMV6) were also purchased from Origene.
Tyagi N., Deshmukh S.K., Srivastava S.K., Azim S., Ahmad A., AL-Ghadhban A., Singh A.P., Carter J.E., Wang B, & Singh S. (2017). ETV4 Facilitates Cell Cycle Progression in Pancreatic Cells through Transcriptional Regulation of Cyclin D1. Molecular cancer research : MCR, 16(2), 187-196.
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4

Cloning and Mutagenesis of URAT1 Genes

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hURAT1 (GenBank BC053348.1), rURAT1 (NCBI NM_001034943.1), and mURAT1 (NCBI NM_009203) genes were purchased from Origene Technologies, Inc. hURAT1 and rURAT1 were subcloned into pCMV6/neo using NotI, creating pCMV6/neo-hURAT1 and pCMV6/neo-rURAT1. Point mutants were produced by polymerase chain reaction or site-directed mutagenesis using the QuikChange Lightning Multi Site Directed Mutagenesis Kit (Agilent Technologies), using mutagenic primers shown in supplementary table S1, Supplementary Material online. All mutants were confirmed by DNA sequencing. Ancestral and baboon URAT1 sequences were synthesized by Blue Heron Biotech, LLC and cloned into pCMV6/neo using EcoRI and XbaI. All URAT1 protein sequences are show in supplementary figure S2, Supplementary Material online.
Tan P.K., Farrar J.E., Gaucher E.A, & Miner J.N. (2016). Coevolution of URAT1 and Uricase during Primate Evolution: Implications for Serum Urate Homeostasis and Gout. Molecular Biology and Evolution, 33(9), 2193-2200.
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