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J. Immunol. 161, 3719-3728. A complex element regulates IFN-gamma-stimulated monocyte chemoattractant protein-1 gene transcription. 1998

Valente, A. J., Xie, J. F. , Abramova, M. A., Wenzel, U. O., Abboud, H. E., Graves, D. T.

Notes: The authors transfected MG-63 human osteoblastic cells with a 10:1 ratio of the MCP-1/firefly luciferase plasmid, derived from the pGL2-Basic Vector, and the pRL-CMV Vector as a control. The calcium phosphate method of transfection was used. Luciferase activities were assayed with the Dual-Luciferase® Reporter Assay System. (0213)

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J. Biol. Chem. 273, 18743-18750. A tripartite array of transcription factor binding sites mediates cAMP induction of phosphoenolpyruvate carboxykinase gene transcription and its inhibition by insulin. 1998

Yeagley, D., Agati, J.M., Quinn, P.G.

Notes: The H4eII cell line was transfected with 20µg of a pGL3 Basic Vector-derived construct, 2µg of various expression vectors and 2µg of the pRL-SV40 Vector. One of the pGL3 Basic Vector-based constructs had the PEPCK promoter plus a GAL4 binding domain and another had five tandem repeats of the GAL4 domain and a minimal TATA box. Fusion proteins of the Kinase-inducible domain and the GAL4 binding domain as well as the Kinase-inducible domain and a cAMP inducible domain. The system was used to measure activation of luciferase via phosphorylation of the GAL4 fusion protein producing a functional activation domain. All transfections and luciferase activities were normalized to Renilla luciferase activity using the Dual-Luciferase® Reporter Assay System. (0111)

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Circulation 98, 1164-1171. Activated platelets induce monocyte chemotactic protein-1 secretion and surface expression of intercellular adhesion molecule-1 on endothelial cells. 1998

Gawaz, M., Neumann, F.-J., Dickfeld, T., Koch, W., Laugwitz, K.-L., Adelsberger, H., Langenbrink, K., Page, S., Neumeier, D., Schömig, A., Brand, K.

Notes: Luciferase reporter studies were performed in primary HUVECs. Experimental construct were prepared in either pGL2 Basic or pGL3 Promoter Vector and cotransfected with the pRL-TK Vector. Reporter activity was monitored with the Dual-Luciferase® Reporter Assay System. (1147)

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J. Immunol. 161, 277-285. An NFAT-dependent enhancer is necessary for anti-IgM-mediated induction of murine CD5 expression in primary splenic B cells. 1998

Berland, R. and Wortis, H.H.

Notes: Reporter assays were performed in murine splenic B cells. Firefly luciferase reporter constructs were contransfected with an equal amount of the pRL-TK Vector. Luciferase activities were assessed with the Dual-Luciferase® Reporter Assay System. (1439)

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J. Biol. Chem. 273, 18751-18759. Assessment of the roles of mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase B and protein kinase C in insulin inhibition of cAMP-induced phosphoenolpyruvate carboxykinase gene transcription. 1998

Agati, J.M., Yeagley, D. and Quinn, P.G.

Notes: Up to four plasmids were transfected into H4IIe cells including 20µg of luciferase reporter vector, 2µg of pRL-SV Vector and an RSV-driven expression vector expressing the catalytic domain of PKA or ras pathway mutants. Luciferase activity was normalized to Renilla luciferase activity using the Dual-Luciferase® Reporter Assay System. (2063)

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Oncogene 16, 423-428. C-Myc 5' untranslated region contains an internal ribosome entry segment. 1998

Stoneley, M., Paulin, F. E., Le Quesne, J. P., Chappell, S. A., Willis, A. E.

Notes: The authors used the Dual-Luciferase® Reporter Assay System, pGL3 Control Vector, pRL-CMV Vector, pSV-beta-Galactosidase Control Vector, Luciferase Assay System and RNase ONE® Ribonuclease in their studies. The objective of the paper was to demonstrate that the 5' untranslated region (UTR) of c-myc functions as an internal ribosome entry site (IRES). To directly assay the ability of the UTR to function as a IRES, the Renilla luciferase gene was subcloned in front of the UTR. Dual expression of the luciferases was demonstrated in both HeLa cells and HepG2 cells and dependent upon the c-myc UTR. Mutation of the Renilla luciferase sequence to contain a hairpin structure, demonstrate that the Renilla luciferase activity could reduced by 75% with no effect on the firefly luciferase activity. RNase protection assays with a 624 nucleotide probe that crossed the Renilla luciferase cDNA, the c-myc UTR and the firefly luciferase cDNA demonstrated that only one single message is produced by the transfected HeLa cells and the c-myc UTR is not functioning as a promoter. All transfections were normalized to control β-galactosidase activity. (0339)

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Immunity 9, 247-256. CD30 is a CD40-inducible molecule that negatively regulates CD40-mediated immunoglobulin class switching in non-antigen-selected human B cells 1998

Cerutti, A., Schaffer, A., Shah, S., Zan, H., Liou, H.-C., Goodwin, R.G., Casali, P.

Notes: Luciferase studies were performed in the human B cell line, CL-01. Cells were electroporated with a 40µl DNA-TE buffer solution containing 20µg of constructs in either pGL3 Basic or pGL3 Promoter Vectors and 10ng of the pRL-CMV Vector. Thus, a 1:2000 ratio of firefly to Renilla luciferase vector is achieved and activity was measured with the Dual-Luciferase® Reporter Assay System. (1353)

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J. Biol. Chem. 273, 27420-27429. Characterization of the muring fatty acid transport protein gene and its insulin response sequence. 1998

Hui, T.Y., Frohnert, B.I., Smith, A.J., Schaffer, J.E., Bernlohr, D.A.

Notes: Luciferase reporter constructs were cotransfected into 3T3-L1 preadipocytes with the pRL-CMV Vector at a 25:1 ratio and luciferase activities were determined with the Dual-Luciferase® Reporter Assay System. RT-PCR was accomplished with AMV Reverse Transcriptase and Taq DNA Polymerase. RNase Protection Assay were performed with the aid of the RNase ONE™ Ribonuclease. (0988)

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Am. J. Physiol. 274, F753-F761. Cis- and trans-acting factors regulating transcription of the BGT1 gene in response to hypertonicity. 1998

Miyakawa, H., Woo, S.K., Chen, C.P., Dahl, S.C., Handler, J.S., Kwon, H.M.

Notes: Use the Dual-Luciferase® Reporter Assay System to study regulatory elements from the BGT1 gene. The vectors used for these studies were the pGL2 Basic and pGL2 Promoter Vectors as primary reporters and the pRL-CMV Vector as a transfection control. (0695)

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J. Biol. Chem. 273, 33885-33888. Cloning and characterization of the 5´-flanking region of the human growth hormone secretagogue receptor gene. 1998

Kaji, H., Tai, S., Okimura, Y., Iguchi, G., Takahashi, Y., Abe, H., Chihara, K.

Notes: Inserted promoter region and its deletion variants into pGL3-Basic Vector and co-transfected with pRL-CMV Vector (no ratio given). Measured luciferase activities with Dual-Luciferase® Reporter Assay System. (0952)

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J. Biol. Chem. 273, 33848-33855. Cloning and characterization of the human β4-integrin gene promoter and enhancers. 1998

Takaoka, A.S., Yamada, T., Gotoh, M., Kanai, Y., Imai, K., Hirohashi, S.

Notes: The authors performed DNaseI footprinting using the Core Footprinting System and nuclear extract or recombinant human AP-1 (c-jun). Promoter and enhancer elements of the human β4 integrin gene were analyzed using the Dual-Luciferase® Reporter Assay System. pRL-TK Vector was used as a transfection control. The transfection ratio of pGL3-Basic-derived plasmids to pRL-TK Vector was 10:1. (0296)

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J. Biol. Chem. 273, 25472–25479. Cloning and characterization of the human PAX2 promoter. 1998

Stayner, C.K., Cunliffe, H.E., Ward, T.A. and Eccles, M.R.

Notes: MDCK and 293 cells were previously shown to express PAX2 endogenously. Transfections were carried out using FuGENE® 6 Transfection Reagent with 10% serum without antibiotics as follows. Cells (COS-7, NIH-3T3, 293 and MDCK cell lines; 5 × 105) were seeded into 60mm dishes and transfected at approximately 50–60% confluency, 16–18 hours later. Cells were harvested 24 hours after transfection. A total of 2.05μg of DNA was used in transfections to analyze the PAX2 promoter deletion series of constructs. These reactions contained 2μg of PAX2 promoter-reporter firefly luciferase plasmid (pGL2-Basic Vector) plus 0.05μg of internal control Renilla luciferase plasmid (pRL-TK Vector). For cotransfection experiments, a total of 4.05μg of DNA was used. The reactions contained 1μg of PAX2 promoter-reporter firefly luciferase plasmid and either 3μg of pBluescript DNA or 3μg of WT1 expression plasmid, plus 0.05μg of internal control Renilla luciferase plasmid (pRL Control Vector).

The DNA solutions were combined at a 1:3 ratio with FuGENE® 6 reagent (e.g. 4.05 μg of DNA to 12 μl of FuGENE® 6 reagent for transfection of each cell line. The cells were overlaid with a final 2ml of medium, for 24 hours prior to lysis. Cell lysates were assayed for luciferase production using a Dual-Luciferase® Assay System.  Luminescence was measured with a luminometer. (4302)

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J. Biol. Chem. 273, 25472-25479. Cloning and characterization of the human PAX2 promoter 1998

Stayner, C.K., Cunliffe, H.E., Ward, T.A., Eccles, M.R.

Notes: Promoter functions were tested in NIH 3T3, COS-7, 293 and MDCK cells. Experimental constructs were prepared in the pGL2 Basic Vector and cotransfected with the pRL-TK Vector at a 40:1 ratio. Luciferase activities were monitored with the Dual-Luciferase® Reporter Assay System. (0332)

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J. Biol. Chem. 273, 7643-7649. Cloning of the multipartite promoter of the sodium-calcium exchanger gene NCX1 and characterization of its activity in vascular smooth muscle cells. 1998

Scheller, T., Kraev, A., Skinner, S., Carafoli, E.

Notes: Promoter activities were examined in primary rat vascular smooth muscle cells. The firefly luciferase vectors were co-transfected with the pRL-TK Vector at a 20:1 ratio. Transfections were performed with an unspecified member of the Tfx™ Reagent trio. (0451)

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J. Immunol. 160, 273. Common and distinct signaling pathways mediate the induction of TNF-alpha and IL-5 in IgE plus antigen-stimulated mast cells. 1998

Csonga, R., Prieschl, E.E., Jaksche, D., Novotny, V., Baumruker, T.

Notes: Luciferase reporter plasmids were cotransfected with pRL-TK Vector at a 5:1 ratio in CPII mouse mast cells. In studies with the reporter vector, ras expression vector and pRL-TK Vector, the ratio of each was 3:5:1 with the amount of pRL-TK Vector constant in all transfections at 2µg per reaction. All transfections were accomplished by electroporation and both luciferase activities were determined with the Dual-Luciferase® Reporter Assay System. (1288)

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Immunity 9, 871-880. Direct suppression of Stat1 function during adenoviral infection. 1998

Look, D.C., Roswit, W.T., Frick, A.G., Gris Alevy, Y., Dickhaus, D.M., Walter, M.J., Holtzman, M.J.

Notes: Used Dual-Luciferase® Reporter Assay System with a 400:1 transfection ratio of the firefly luciferase plasmid to pRL-SV40 Vector. Also used the pCI-neo Mammalian Expression Vector to make several E1A expression plasmids. (0743)

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J. Biol. Chem. 273, 26969-26976. Down-regulation of cytochrome P450 1A1 gene promoter by oxidative stress: Critical contribution of nuclear factor 1. 1998

Morel, Y., Barouki, R.

Notes: Promoter activities were studied in HepG2 cells with the Dual-Luciferase® Reporter Assay System. The Renilla control vector was constructed from the pRL-SV Vector by replacing the SV40 promoter with the α-globin gene proximal promoter (–79-+1). The Firefly luciferase construct was prepared in the pGL3 Basic Vector. The pGL3-based Vector and pRL-based Vector were transfected at a 3:1 ratio. A mutant nuclear factor 1 site was introduced into the 1A1 promoter with the GeneEditor™ in vitro Site-Directed Mutagenesis System by converting GCCA to CGCA. (0659)

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Alcohol Clin. Exp. Res. 22, 849-853. Ethanol transcriptionally upregulates t-PA and u-PA gene expression in cultered human endothelial cells. 1998

Grenett, H.E., Aikens, M.L., Torres, J.A., Denissle, S., Tabengwa, E.M., Davis, G.C., Booyse, F.M.

Notes: Studies were performed in low passage HUVEC cells. Two micrograms of pGL2 Basic-derived construct was cotransfected with 0.15µg of the pRL-TK Vector (13:1 ratio). The luciferase activity was determined with the Dual-Luciferase® Reporter Assay System. (1120)

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Mol. Cell 1, 905-911. Functional analysis of the human TAFII250 N-terminal kinase domain 1998

O'Brien, T., Tijan, R.

Notes: Studies were performed in ts13 cells that have a temperature-sensitive TAFII250. Either cyclin A or cdc2 luciferase vectors were cotransfected with wildtype or mutant TAFII250 proteins as well as pRL-CMV Vector (luciferase vector: Renilla vector; 20:1). Luciferase activity was measured with the Dual-Luciferase® Reporter Assay System (0586)

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J. Immunol. 161, 3822. GATA-3-dependent enhancer activity in IL-4 gene regulation. 1998

Ranganath, S., Ouyang, W., Bhattarcharya, D., Sha, W.C., Grupe, A., Peltz, G., Murphy, K.M.

Notes: Three plasmids were transfected into M12 cells or Jurkat cells: Cytokine promoter-driven firefly luciferase reporter vector; GATA-3 expression vector and pRL-CMV Vector. The vectors were electroporated at a 40:40:1 ratio. Luciferase activity was determined with the Dual-Luciferase® Reporter Assay System. (0497)

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J. Biol. Chem. 273, 9457-9464. Genomic organization, chromosomal mapping, and promoter analysis of the mouse dentin sialophophoprotein (Dspp) gene, which codes for both dentin sialoprotein and dentin phosphoprotein. 1998

Feng, J.Q., Luan, X., Wallace, J., Jing, D., Ohshima, T., Kulkarni, A.B., D'Souza, R.N., Kozak, C.A., MacDougal, M.

Notes: Reporter assays were performed in the odontoblast cell line, MO6-G3. Experimental promoter constructs were assembled in the pGL3 Basic Vector and transfections were control through the use of the pRL-SV Vector. Luciferase activity was measured with the Dual-Luciferase® Reporter Assay System. Primer extension analysis of the gene transcript was performed with the Primer Extension System. (1196)

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J. Biol. Chem. 273, 25466-25471. Heat shock factor 1 mediates hemin-induced hsp70 gene transcription in K562 erythroleukemia cells 1998

Yoshima, T., Yura, T., Yanagi, H.

Notes: Reporter studies were performed in K562 cells. The Dual-Luciferase® Reporter Assay System was used to measure the firefly luciferase from a GAL4 binding site-containing luciferase vector and cotransformed pRL-SV40 Vector. No ratios were reported. Other vectors were transfected as well and produced proteins that activated that bound the GAL4 domain and activated transcription of the firefly luciferase in a two-hybrid-type assay. Another vector was constructed with Heat Shock Elements instead of GAL4 domains in a pGL3 Promoter Vector. (0116)

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J. Biol. Chem. 273, 33741. Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins: Involvement of basic leucine zipper transcription factors. 1998

Yoshida, H., Haze, K., Yanagi, H., Yura, T., Mori, K.

Notes: Promoter studies were performed in HeLa cells. The promoter constructs were assembled in the pGL3 Basic Vector (10µg) and cotransformed with the pRL-SV40 Vector (0.1µg) at a 10:1 ratio using the calcium phosphate method. Reporter activity was measured with the Dual-Luciferase® Reporter Assay System. The TNT® T7 Quick Coupled Transcription/Translation System was used to produce a positive control protein for Western blot analysis of transfected HeLa cell extracts. (0115)

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Am. J. Physiol. 274, C681-C687. In vivo analysis of the myosin heavy chain IIB promoter region. 1998

Swoap, S. J.

Notes: The authors cloned a 2.6kb promoter-enhancer region of the MHC IIB gene into the pGL3-Basic Vector. They injected this construct along with the pRL-CMV Vector into mouse muscle cells. Tissue extracts were prepared with a buffered Tris solution containing a protease inhibitor cocktail and assayed for reporter activity using the Dual-Luciferase® Reporter Assay System. (0287)

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J. Biol. Chem. 273, 22120-22127. Induction of cyclooxygenase-2 by activated ha-ras oncogene in Rat-1 fibroblasts and the role of mitogen-activated protein kinase pathway. 1998

Sheng, H., Williams, C.S., Shao, J., Liang, P., DuBois, R.N., Beauchamp, R.D.

Notes: Luciferase studies were performed in a Rat-1 fibroblast cell line with an inducible Ha-ras gene. Experimental pGL3 Basic vectors were cotransfected with pRL-TK at a 2:1 ratio. (0389)

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