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J. Biol. Chem. April 28, ePub ahead of print. Mitogenic responses of vascular smooth muscle cells to lipid peroxidation-derived aldehyde 4-hydroxy-trans-2-nonenal (HNE): Role of aldose reductase-catalyzed reduction of the HNE-glutathione conjugates in regulating cell growth. 2006

Ramana, K.V., Bhatnagar, A., Srivastava, S., Umesh C.S. Yadav, Awasthi, S., Awasthi, Y.C. and Srivastava, S.K.

Notes: Protein Kinase C activity in rat vascular smooth muscle cells in response aldose reductase was measured using the SignaTECT® Protein Kinase C (PKC) Assay System. Expression of NFκB and AP-1 was investigated by electrophoretic mobility gel shift assays using the AP-1 and NFκB Consensus Oligonucleotides. (3406)

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Mol. Cell. Biol. 24(5), 2169-80. A transforming growth factor beta-induced Smad3/Smad4 complex directly activates protein kinase A. 2004

Zhang, L., Duan, C.J., Binkley, C., Li, G., Uhler, M.D., Logsdon, C.D. and Simeone, D.M.

Notes: These authors investigated the possible interaction between the TGFß and PKA signaling pathways. Mv1Lu (fetal mink lung) cells or pancreatic acini from male Swiss Webster mice were treated with TGFß1, washed and protein extracted.  After determining concentration, equal amounts of protein were used in the SignaTECT® cAMP-Dependent Protein Kinase (PKA) Assay System. The CREB Consensus Oligonucleotide was used in gel shift and supershift assays with nuclear extract prepared from Mv1Lu cells treated with increasing concentrations of TGFß1 to demonstrate the effect of the growth factor on DNA binding.  Cell proliferation was measured using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay. (3114)

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J. Biol. Chem. 277(21), 18411-20. Kaurane diterpene, kamebakaurin, inhibits NF-kappa B by directly targeting the DNA-binding activity of p50 and blocks the expression of antiapoptotic NF-kappa B target genes. 2002

Lee, J.H., Koo, T.H., Hwang, B.Y. and Lee, J.J.

Notes: To investigate the effect of the compound kamebakaurin (KA) on NF-κB, an NF-κB-responsive firefly luciferase vector was transfected into HeLa, Jurkat and THP-1 cells. The Luciferase Assay System was used to assay the level of NF-κB induction after treatment of cells with various concentrations of KA. To determine if KA influenced the DNA-binding activity of NF-κB, nuclear extracts of HeLa, Jurkat and THP-1 cells were prepared after preincubation with KA and stimulation of NF-κB activity. Control nuclear extracts were prepared from unstimulated p50- or RelA-overexpressed MCF-7 cells. In addition, the wildtype and DNA-binding mutant RelA and p50 (NF-κB) His-tagged proteins were translated using the TNT® Quick Coupled Transcription/Translation System and subsequently purified. Using the Gel Shift Assay System, the NF-κB and AP1 oligos were tested for electromobility shifts with the prepared nuclear extracts or with purified wildtype and mutant proteins. Supershift studies using anti-p50 or anti-RelA antibodies were also performed. (3113)

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Nucl. Acids Res. 29, 21. Development of a sensitive multi-well colorimetric assay for active NF-κB. 2001

Renard, P., Ernest, I., Houbion, A., Art, M., Le Calvez, H., Raes, M. and Remacle, J.

Notes: A double-stranded NF-κB consensus oligonucleotide sequence containing 122bp oligonucleotide was created by PCR with a biotinylated primer at the 5´ end and the NF-κB Consensus Oligonucleotide sequence at the 3´ end. The resultant probe was bound to streptavidin-coated plates and used to screen for activated NF-κB in IL-1β-stimulated WI-38 VA13 human fibroblast cell lysates. Actively bound NF-κB was detected colorimetrically with either a rabbit anti NF-kB p50 or p65 antibody, followed by a goat anti-rabbit HRP conjugated secondary antibody and a TMB solution. Recombinant hNF-κB (p50) was used as a standard in the assay. The authors provide details for each step in the process including the specific buffers, lysate volumes, and incubation times and temperatures. The NF-κB Consensus Oligonucleotide was also used in EMSAs to confirm the results observed in the screening assay. For this assay the authors used 25μg of total protein from each lysate preparation. (2690)

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J. Biol. Chem. 273, 6607-6610. IκBα degradation and nuclear factor-κB DNA binding are insufficient for interleukin-1β and tumor necrosis factor-α-induced κB-dependent transcription. Requirement for an additional activation pathway. 1998

Bergmann, M., Hart, L., Lindsay, M., Barnes, P.J. and Newton, R.

Notes: IL-1β and TNFα  have been shown to induce two p50/p65 NF-κB DNA binding complexes in A549 cells. Tfx™-50 Reagent was used to stably transfect A549 cells in order to study the effects on NF-κB-dependent transcription. Promega's Luciferase Assay System was used to measure the effect of kinase inhibitors on IL-1β and TNFα stimulated NF-κB-dependent transcription. In addition, Promega's NF-κB and OCT1 Oligonucleotides were used in gel shift assays (1438)

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Arterioscler. Thromb. Vasc. Biol. 18, 473-480. LDL induces transcription factor activator protein-1 in human endothelial cells. 1998

Zhu, Y., Lin, J.H.-C., Liao, H.-L., Friedli, O.Jr., Verna, L., Marten, N.W., Straus, D.S. and Stemerman, M.B.

Notes: Consensus Oligonucleotides for NF-κB, AP-1 and CREB were used for gel shift assays with nuclear extracts from HUVEC cells. A basal reporter was constructed in the pGL2 Basic Vector by subcloning the TATA box of the HSV TK promoter. To make an AP-1 responsive reporter, the AP-1 Consensus Oligonucleotide was phosphorylated and ligated into the TATA vector at a 120:1 ratio. A clone with three tandem repeats of the AP-1 consensus site was identified by sequencing. Transfections into COS-7 cells were performed using the ProFection® Mammalian Transfection System-DEAE Dextran. (0067)

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J. Biol. Chem. 273, 21594-21602. Molecular cloning and characterization of a transcription regulator with homology to GC-binding factor 1998

Reed, A.L., Yamazaki, H., Kaufman, J.D., Rubinstein, Y., Murphy, B., Johnson, A.C.

Notes: The cloned 752 amino acid GC-binding factor 2 (GCF2) was expressed in vitro using the TNT® Coupled Reticulocyte Lysate System. The synthesized product migrated at ~160kDa by SDS-PAGE when the predicted molecular weight was 83kDa. Expression of the protein from E.coli also produced a protein that migrated at ~160kDa. Mass spectrum analysis confirm a mass of 83kDa. The E.coli produced protein was further characterized by gel shift analysis with the AP-2 Consensus Oligonucleotide. Recombinant GCF2, AP-2 and SP1 transcription factors were analyzed for binding to the EGF receptor promoter by footprint analysis. The GCF2 protein was expressed with a EGF Receptor promoter-CAT reporter vector in CV-1 cells and found to increase CAT activity. (0510)

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J. Biol. Chem. 273, 32787-32792. Protein Kinase C-alpha modulates lipopolysaccharide-induced functions in a murine macrophage cell line. 1998

St Denis, A., Chano, F., Tremblay, P., St Pierre, Y., and Descoteaux, A.

Notes: The authors examined the effect of overexpressing a dominant negative mutant of protein kinase C alpha in RAW 264.7 cells on LPS induced signal transduction. They used the Anti-ACTIVE® JNK pAb for western analysis and the NF-kappaB oligonucleotide for electrophoretic mobility shift assay (EMSA) analysis of NF-kappaB activation. (0326)

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Am. J. Physiol. 274, L289-L295. Regulation of surfactant proteins A and B by TNF-alpha and phorbol ester independent of NF-kappa B. 1998

Pryhuber, G.S., Khalak, R., Zhao, Q.

Notes: The Promega NF-kappa B Consensus Oligonucleotide and HeLa Nuclear Extract were used in gel-shift experiments. (0518)

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J. Immunol. 160, 4353-4360. Regulation of transcription of the TATA-less human complement component C4 gene. 1998

Vaishnaw, A. K. , Mitchell, T. J. , Rose, S. J. , Walport, M. J. , Morley, B. J.

Notes: The authors used the Primer Extension System - AMV reverse transcriptase to determine transcription efficiencies of transfected constructs. Also, the fmol® DNA Cycle Sequencing System was used to confirm the nature of the constructs. EMSAs were carried out using Promega's SP1 Oligonucleotides. (0212)

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J. Biol. Chem. 273, 14885-14890. Transcriptional regulation of endothelial nitric-oxide synthase by lysophosphatidylcholine 1998

Cieslik, K., Zembowicz, A., Tang, J.-L., Wu, K.K.

Notes: The Serine/Threonine Phosphatase Assay System was used to assess protein phosphatase 2A, 2B and 2C activity in nuclear extracts of Human umbilical vein cells (HUVEC) with or without lysophosphatidylcholine treatment. Gel shifts were also performed with nuclear extracts prepared from the HUVEC cells using the SP1 Consensus Oligonucleotide. Luciferase reporter studies were also performed in HUVEC cells using pGL3 Basic-derived vectors and the Luciferase Assay System. (1305)

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J. Biol. Chem. 272, 16934-16939. Activating transcription factor 2 (ATF2) down-regulates hepatitis B virus X promoter activity by the competition for the activating protein 1 binding site and the formation of the ATF2-Jun heterodimer 1997

Choi, C. Y. , Choi, B. H. , Park, G. T. , Rho, H. M.

Notes: The AP-1 and CREB Consensus Oligonucleotides were used for gel shift assays with transfected HepG2 cell extracts, E.coli extracts containing eukaryotic transcription factors and a truncated c-jun protein expressed with Rabbit Reticulocyte Lysate. (1343)

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J. Biol. Chem. 272, 14914-14920.. Activation of NF-kappaB by antineoplastic agents. Role of protein kinase C. 1997

Das, K. C. , White, C. W.

Notes: Gel Shifts performed in A549 adenocarcinoma nuclear extracts using the AP1, AP2, SP1, CREB, TFIID, and NF-kappaB Consensus Oligonucleotides and the Gel Shift Assay System. (1255)

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Proc. Natl. Acad. Sci. USA 94, 9487-9492. Characterization of the α1B-adrenergic receptor gene promoter region and hypoxia regulatory elements in vascular smooth muscle. 1997

Eckhart, A.D. , Yang, N. , Xin, X. , Faber, J.E.

Notes: Luciferase studies were performed in aortic and vena caval smooth muscle cells. Transfections were normalized to β-galactosidase activity. Researchers report that pGL3 Promoter and pGL3 Control Vectors were 55-± 3-fold and 340±41-fold greater activity, respectively, than the pGL3 Basic Vector alone. The CREB consensus oligo was used in gel shifts of aortic and vena cava smooth muscle cell nuclear extracts. (1214)

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J. Neurosci. 17, 6554-6564.. Differential expression of alpha-bungarotoxin-sensitive neuronal nicotinic receptors in adrenergic chromaffin cells: A role for transcription factor Egr-1. 1997

Criado, M., Dominguez del Toro, E.D., Carrasco-Serrano, C., Smillie, F.I., Juíz, J.M., Viniegra, S. and Ballesta, J.J.

Notes: Total RNA was isolated from adrenomedullary tissue dissected from areas near to or far from the adrenal cortex. The isolated RNA was used for RT-PCR. Reporter studies were performed in Neuro2a murine neuroblastomas, SH-SY5Y human neuroblastomas and bovine chromaffin cells. All luciferase activities were normalized to control beta-Galactosidase activity. The consensus oligonucleotides were used in gel shift assays of bovine chromaffin cell nuclear extracts. The oligonucleotides were used to show specificity for Egr-1. (1614)

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J. Biol. Chem. 272, 24624-24630. Failure of a second X-ray dose to activate nuclear factor κB in normal rat astrocytes. 1997

Raju, U., Lu, R., Noel, F., Gumin, G.J. and Tofilon, P.J.

Notes: The authors examined gel shifts of the NF-κB consensus oligonucleotide by nuclear extracts of primary rat astrocytes that were exposed to various amounts of X-rays. Supershifts were also performed. Promega's Gel Shift Assay System and NF-κB consensus oligonucleotide were used. (1523)

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J. Biol. Chem. 272, 9648-9654. Integration of Jak-Stat and AP-1 signaling pathways at the vasoactive intestinal peptide cytokine response element regulates ciliary neurotrophic factor-dependent transcription. 1997

Symes, A., Gearan, T., Eby, J. , Fink, J.S.

Notes: AP1 Consensus Oligonucleotide was used in gel shift assays with NBFL cell extracts. (0289)

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J. Biol. Chem. 272, 5936-5942. Nerve growth factor up-regulates the N-methyl-D-aspartate receptor subunit 1 promoter in PC12 cells. 1997

Bai, G. and Kusiak, J.W.

Notes: Reporter vectors (produced with the pGL2 Basic Vector) were transiently transfected into PC12 cells and promoter activity was measured in response to 2.5S NGF. Promoter constructs were generated by PCR, subcloned into the pGEM®-T Vector and sequenced. The pGEM®-luc Vector was used to generated an antisense luc RNA probe used in RNase protection assays (using RNase ONE™ Ribonuclease) to follow the transcription of luciferase DNA following NGF treatment. The early growth reaction transcription factor and variants were expressed in the TNT® SP6 Coupled Reticulocyte Lysate System and used to perform gel shifts with promoter elements. The SP1 Consensus Oligonucleotide was used to compete transcription factor binding to the promoter elements. (1493)

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Am. J. Physiol. 273, F507-F515. PGE2-mediated cytoprotection in renal epithelial cells: evidence for a pharmacologically distinct receptor. 1997

Weber, T. J. , Monks, T. J. , Lau, S. S.

Notes: The authors used the AP1 and AP2 Consensus Oligonucleotides in their studies. (0175)

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J. Biol. Chem. 272, 18990-18999. Specific Activation of Retinoic Acid Receptors (RARs) and Retinoid X Receptors Reveals a Unique Role for RARgamma in Induction of Differentiation and Apoptosis of S91 Melanoma Cells 1997

Spanjaard, R. A., Ikeda, M., Lee, P. J., Charpentier, B., Chin, W. W., Eberlein, T. J.

Notes: The CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS/PMS) was used to measure the proliferation of S91 cells. The TNT® Coupled Reticulocyte Lysate System was used to translate the retinoic acid receptor and the retinoid X receptor. These were then assayed in gel shifts. The recombinant human AP-1 and the AP-1 Consensus Oligonucleotides were used in the gel shift assays as well. (0360)

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J. Biol. Chem. 272, 14244-14250. Transcription factor AP-2 controls transcription of the human transforming growth factor-alpha gene. 1997

Wang, D., Shin, T. H., Kudlow, J. E.

Notes: Luciferase studies were performed with MDA468 human mammary carcinoma cells using constructs prepared in the pGL2-Basic Vector. Gel shift assays were performed with extracts of these cells using the AP2 and SP1 Consensus Oligonucleotides. (0197)

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Blood 89, 2891-2900. Two distinct pathways of interleukin-5 synthesis in allergen-specific human T-cell clones are suppressed by glucocorticoids. 1997

Mori, A., Kaminuma, O., Suko, M., Inoue, S., Ohmura, T., Hoshino, A., Asakura, Y., Miyazawa, K., Yokota, T., Okumura, Y., Ito, K., Okudaira, H.

Notes: Luciferase reporter studies were performed in antigen-specific T cell clones. Experimental constructs were prepared in the pGL2 Basic Vector and luciferase activities were monitored with the Luciferase Assay System. Nuclear extracts were then made from these T cell clones and used for gel shifts with the Gel Shift Assay System. The analysis included the use of the AP-1 Consensus Oligonucleotide, the NF-kappaB Consensus Oligonucleotide and the Oct-1 Consensus Oligonucleotide. (0661)

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J. Biol. Chem. 272, 6051-6058. Tyrosine hydroxylase gene promoter activity is regulated by both cyclic AMP-responsive element and AP1 sites following calcium influx. Evidence for cyclic amp-responsive element binding protein-independent regulation. 1997

Nagamoto-Combs, K., Piech, K.M., Best, J.A., Sun, B., Tank, A.W.

Notes: The pCAT® Basic Vector was used to measure baseline CAT activity in PC12 cells. The Sp1 Consensus Oligonucleotide was used for gel shifts from transfected PC12 cells. (The pCAT® Basic Vector has been replaced by the next generation vector, pCAT®3 Basic.) (0640)

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Anal. Biochem. 213, 162-167. Enhanced gel mobility shift assay for DNA-binding factors. 1993

Hassanain, H.H., Dai, W., Gupta, S.L.

Notes: The authors used AP1 and SP1 Consensus Oligonucleotides in an improved binding buffer containing detergents. (1068)

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