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| 1. |
Wilson, P.M., Fazzone, W., LaBonte, M.J., Lenz, H.J. and Ladner, R.D.
(2009)
Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage.
Nucleic Acids Res.
37
,
78–95
.
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Notes:
The authors examined the role of p53 in modulating dUTPase promoter activity. Base substitution mutations of Sp1- and E2F-binding sites in the dUTPase promoter were performed using the GeneEditor™ in vitro Site-Directed Mutagenesis System. Each mutant was confirmed by DNA sequencing. To determine growth inhibition, HCT116 human colon cancer cells were seeded in 96-well plates at 3 × 103 cells/well and treated with 5-fluorouracil (5-FU), fluorodeoxyuridine (FUdR), oxaliplatin or in combination. After 72 hours, the CellTiter® 96 AQueous One Solution was dispensed into each well and absorbance measured. RNA was isolated from HCT116 p53+/+ and HCT116 p53-/- cells. cDNA was reverse transcribed from 200ng total RNA followed by multiplex qPCR using the Plexor™ qPCR System to amplify dUTPase, thymidylate synthase and GAPDH, a housekeeping gene. The 1.2 kb region of the dUTPase promoter upstream of the transcriptional start site was amplified by PCR and the fragment cloned into the pGL3-Basic Vector. Truncated promoters were also generated by PCR and cloned into the same vector. Drosophila SL-2 cells and HCT116 cell lines were seeded in a 24-well plate and transfected with dUTPase pGL3 promoter constructs or with pCI-Neo:p53WT, pCI-Neo:p53MUT and the empty pCI-neo Mammalian Expression Vector; all transfections included the pRL-TK Vector at a ratio of 1:10. After six hours, the cells were incubated in either fresh medium or medium containing a cytotoxic agent at the appropriate concentration. Thirty hours later, the cells were lysed, quantitated by Western blotting and 20µl of lysate analyzed with the Dual-Luciferase® Reporter Assay System. Electrophoretic mobility shift analyses (EMSA) were performed using –64 to –91 of the dUTPase-nuclear isoform transcriptional start site in the Gel Shift Assay System.
(0004031) |
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Products: CellTiter 96® AQueous One Solution Cell Proliferation Assay | CellTiter 96® AQueous One Solution Reagent | CellTiter 96® AQueous One Solution Reagent | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | Gel Shift Assay Core System | Gel Shift Assay System | GeneEditor™ in vitro Site-Directed Mutagenesis System | pCI Mammalian Expression Vector | pGL3 Basic Vector | Plexor® qPCR System | pRL-TK Vector |
| 2. |
Haraguchi, M., Okubo, T., Miyashita, Y., Miyamoto, Y., Hayashi, M., Crotti, T.N., McHugh, K.P. and Ozawa, M.
(2008)
Snail regulates cell-matrix adhesion by regulation of the expression of integrins and basement membrane proteins.
J. Biol. Chem.
283
,
23514–23
.
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Notes:
Snail is a transcriptional repressor of E-cadherin that enhances both cell attachment and cell detachment in Madin Darby canine kidney (MDCK) and A4231 cells. To investigate this effect, the authors used Western blot analysis and RT-PCR to monitor protein and mRNA levels of the major adhesive proteins expressed in epithelial cells: laminin, heparin sulfate proteoglycan and collagens. For RT-PCR, total RNA was isolated from transiently transfected snail-expressing MDCK and A431 cells and untransfected cells, then reverse transcribed. The resulting cDNA was amplified by PCR using GoTaq® DNA Polymerase; glyceraldehyde-3-phosphate dehydrogenase was amplified as an internal control. The ability of Snail to regulate the integrin αV promoter was also examined by cloning the promoter and several promoter deletions upstream of a firefly luciferase reporter gene in the pGL3-Basic Vector. Each of these constructs (1µg) and 20ng of pRL-CMV Vector were transfected into MDCK and MDCK/snail cells, and luminescence was measured using the Dual Luciferase Assay System.
(0003882) |
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Products: Dual-Luciferase® Reporter Assay System | GoTaq® DNA Polymerase | pGL3-Basic Vector |
| 3. |
Cali, J.J., Niles, A., Valley, M.P., O’Brien, M.A., Riss, T.L., and Shultz, J.
(2008)
Bioluminescent assays for ADMET
Expert Opin. Drug Metab. Toxicol.
4
,
103–120
.
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Notes:
The authors of this review article highlight the use of bioluminescence as a readout for high-throughput ADME/Tox assays. They discuss three strategies for designing bioluminescent assays, using either luciferase, ATP or luciferin substrates as the limiting reagents for a luciferase-catalyzed reaction. Reporter gene assays limit the production of luciferase by tying it to a promoter or DNA regulatory region of interest. Such assays can be used to study genes that are regulated by drugs and other xenobiotics. Bioluminescent assays in which ATP is the limiting reagent of the luciferase reaction can be designed to monitor cell viability or the activity kinases. Bioluminescent assays in which the substrate is limiting can be designed so that the activity of a particular enzyme results in the production of a luciferin substrate that can, in turn, be acted upon by luciferase.
(0003926) |
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Products: Apo-ONE® Homogeneous Caspase-3/7 Assay | Apo-ONE® Homogeneous Caspase-3/7 Buffer | Beta-Glo® Assay System | Calpain-Glo™ Protease Assay | Caspase-Glo® 2 Assay | Caspase-Glo® 3/7 Assay | Caspase-Glo® 6 Assay | Caspase-Glo® 8 Assay | Caspase-Glo® 9 Assay | CellTiter-Glo® Luminescent Cell Viability Assay | GloResponse™ CRE-luc2P HEK293 Cell Line | GloResponse™ NFAT-RE-luc2P HEK293 Cell Line | GSH-Glo™ Glutathione Assay | Kinase-Glo® Luminescent Kinase Assay | Kinase-Glo® Max Luminescent Kinase Assay | Kinas |
| 4. |
Heikkinen, L.S., Kazlauskas, A., Melén, K., Wagner, R., Ziegler, T., Julkunen, I. and Saksela, K.
(2008)
Avian and 1918 Spanish influenza a virus NS1 proteins bind to Crk/CrkL Src homology 3 domains to activate host cell signaling.
J. Biol. Chem.
283
,
5719–27
.
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Notes:
The authors identified the N-terminal Src homology 3 (SH3) domain-binding motif of Crk and CrkL as the preferred binding partner of nonstructural protein 1 (NS1), an important virulence factor of the influenza A virus. Interaction of NS1 with Crk, CrkL and other SH3 domain-containing proteins p85, p85β and Eps8L1 was investigated by protein pull-down assays. Expression constructs for biotinylated Crk, CrkL, p85, p85β and Eps8L1 were created by amplifying the 123 amino acid biotin acceptor domain from Propionibacterium shermanii from the PinPoint Xa-T Vector and inserting it upstream of the protein-coding sequences in a pGEX vector derivative. These constructs and a construct encoding Myc-tagged NS1 were transfecting into 293FT cells, and the biotinylated proteins were immobilized from the cell lysate using TetraLink™ Tetrameric Avidin Resin. Any Myc-NS1 that bound to the immobilized protein was detected using Western Blot analysis and an anti-Myc antibody. The authors also investigated the ability of wildtype NS1 or NS1 mutants to inhibit interferon-induced gene expression. A reporter plasmid was created by cloning an interferon-stimulated response element upstream of a minimal thymidine kinase promoter driving firefly luciferase expression. A vector containing Renilla luciferase was used as a control to normalize transfection efficiency. Huh-7 cells were cotransfected with the firefly and Renilla luciferase reporter constructs (0.2µg and 5ng, respectively), treated with interferon-β and lysed using the Passive Lysis Buffer. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay System.
(0003803) |
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Products: Dual-Luciferase® Reporter Assay System | TetraLink™ Tetrameric Avidin Resin |
| 5. |
Birdsey GM, Dryden NH, Amsellem V, Gebhardt F, Sahnan K, Haskard DO, Dejana E, Mason JC, Randi AM.
(2008)
Transcription factor Erg regulates angiogenesis and endothelial apoptosis through VE-cadherin.
Blood
111
,
33498-33506
.
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Notes:
These authors showed that the ETS transcription factor Erg interacts with the VE-cadherin promoter region and regulates endothelial apoptosis through this interaction. They demonstrated that inhibition of Erg by siRNA resulted in decreased VE-cadherin mRNA and protein levels, and showed that Erg interacts with the VE-cadherin promoter using a CHIP assay. To show the functional relevance of this interaction, HeLa cells were transfected with a pGL4 Vector containing the VE-cadherin promoter region and an expression vector containing Erg2 cDNA. In this reporter assay, Erg overexpression resulted in ~1.8 fold transactivation of VE-cadherin promoter activity, as measured using the Dual-Luciferase® Reporter Assay System. Inhibition of Erg in human umbilical vein endothelial cells also resulted in a loss of viability and an increase in activation of caspase 3 and caspase 7. The authors showed that apoptosis could be significantly decreased by overexpression of VE-cadherin, indicating that Erg regulates survival partially via its interaction with VE-cadherin. The Caspase-Glo® 3/7 Assay was used to measure caspase activity in these experiments.
(0003872) |
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Products: Caspase-Glo® 3/7 Assay | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack |
| 6. |
Cavallaro, M., Mariani, J., Lancini, C., Latorre, E., Caccia, R., Gullo, F., Valotta, M., DeBiasi, S., Spinardi, L., Ronchi, A., Wanke, E., Brunelli, S., Favaro, r., Ottolenghi, S. and Nicolis, S.K.
(2008)
Impaired generation of mature neurons by neural stem cells from hypomorphic Sox2 mutants
Development
135
,
541-557
.
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Notes:
The authors of this paper investigated the role of Sox2 in neuronal differentiation. Neurospheres were derived from the brains of normal and Sox2 hyphomorphic mice and used to generate differentiated neural cells. In astroglia from cultures containing a Sox2-GFP-expressing lentivirus, ectopic expression of Sox2 correlated with reduced GFAP expression. The authors investigated the role of Sox2 by amplifying binding sites upstream of the GFAP promoter and cloning them upstream of the thymidine kinase promoter in the pRL-TK vector. The Dual-Luciferase Assay System was used to analyze the effect of Sox2 expression on the luciferase reporter gene. The DeadEnd™ Fluorometric TUNEL System was also used to assess apoptosis in some of the neurosphere-derived cultures.
(0003953) |
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Products: DeadEnd™ Fluorometric TUNEL System | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | pRL-TK Vector |
| 7. |
Dekoter, R.P., Schweitzer, B.L., Kamath, M.B., Jones, D., Tagoh, H., Bonifer, C., Hildeman, D.A., and Huang, K.J.
(2007)
Regulation of the interleukin-7 receptor α-promoter by the Ets transcription factors PU.1 and GA-binding protein in developing B cells.
J. Biol. Chem.
282
,
14194-14204
.
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Notes:
The interleukin-7 receptor is composed of γ and α subunits, encoded by the genes il7rg and il7r, respectively. The α subunit is expressed in developing B cells and is downregulated upon maturation. These authors investigated the mechanisms of transcriptional regulation of the il7r gene using 5´ RACE, EMSA, RNA interference and chromatin immunoprecipitation analyses. Potential promoter regions identified by 5´ RACE analysis were cloned into the pGL3-Basic luciferase reporter vector for further study. The promoter constructs were transiently transfected into the 38B9 pro-B cell line along with the control pRL-TK Vector, which expresses Renilla luciferase, and the Dual-Luciferase® Reporter Assay System was used to assess luciferase activity from the various promoter constructs. The promoter construct having the highest activity was chosen, and site directed mutagenesis was used to identify specific regions within the promoter fragment that may be important for activity. Sequence analysis was then used to identify a conserved Ets transcription factor binding site within the putative il7r promoter region. To determine whether the ETS transcription factor GABP binds to this Ets region, the authors performed chromatin immunoprecipitation analysis with an anti-GABP antibody. Immunoprecipitated DNA was then PCR-amplified with primers specific for the Ets region or control primers. The Wizard® SV Gel and PCR Clean-Up System was used to purify the amplified fragments prior to semiquantitative PCR analysis.
(0003626) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pGL3-Basic Vector | pRL-TK Vector | Wizard® SV Gel and PCR Clean-Up System |
| 8. |
Polesskaya, A., Cuvellier, S., Naguibneva, I., Duquet, A., Moss, E.G. and Harel-Bellan, A.
(2007)
Lin-28 binds IGF-2 mRNA and participates in skeletal myogenesis by increasing translation efficiency.
Genes Dev.
21
,
1125–1138
.
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Notes:
The protein Lin-28 is a translational enhancer in differentiating myoblasts; one target of Lin-28 is insulin-like growth factor 2 (IGF-2). The authors showed that Lin-28 increased expression of an IGF-2 reporter construct in vitro. [35S]-methionine-labeled, His-tagged Lin-28 was expressed in the TNT® Coupled Reticulocyte Lysate System and purified using the MagZ™ Protein Purification System. Increasing amounts of purified Lin-28 protein were added to a TNT® Coupled Reticulocyte Lysate System reaction containing an IGF-2 luciferase reporter vector, and as a result, luciferase expression was increased up to threefold. Experiments performed with an irrelevant His-tagged protein of equal size and with an equal number of methionine residues confirmed that the increase in luciferase activity was specific to Lin-28. Side-by-side experiments performed with a luciferase reporter vector without the IGF-2 regulatory element did not show increased luciferase activity.
(0003717) |
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Products: MagZ™ Protein Purification System | TNT® T7 Coupled Reticulocyte Lysate System |
| 9. |
Tan, K.P., Yang, M. and Ito, S.
(2007)
Activation of Nrf2 by toxic bile acids provokes adaptive defense responses to enhance cell survival at the emergence of oxidative stress.
Mol. Pharmacol.
72
,
1380–1390
.
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Notes:
The authors explored the role that nuclear factor (erythroid 2-like) factor 2 (Nrf2) may have in mitigating the cytotoxic effects of bile acids on cells. A reporter vector was constructed using the core sequence of antioxidant reporter element (ARE) sythnesized by annealing two complementary oligonucleotides with Kpn1 and BglII at the 5’ and 3’ ends, respectively, and ligated into the same restriction sites of the pGL3-Promoter Vector (designated pGL3_ARE). To ensure specificity for the experiments, three point mutations were introduced into the ARE sequence (designated pGL3_mARE). To create HepG2 cells that stably expressed human Na(+)-taurocholate co-transporting polypeptide (NTCP), the cDNA clone of NTCP was subcloned into pTargeT™ Mammalian Expression Vector via the NotI site and selected using 500μg/ml G-418. The stable clones or standard HepG2 cells were transiently transfected with 0.1µg of pGL3_ARE or pGL3_mARE, 0.02µg of the control reporter pRL-TK Vector, with or without Nrf2 or dominant negative Nrf2 expression constructs. After overnight transfection, the cells were treated with bile acids for 16–18 hours and luciferase activities determined using the Dual-Luciferase® Reporter Assay System. Each experiment was done in triplicate and repeated at least two times.
(0003691) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pGL3-Promoter Vector | pRL-TK Vector | pTARGET™ Mammalian Expression Vector System |
| 10. |
Igarashi, M., Yogiashi, Y., Mihara, M., Takada, I., Kitagawa, H. and Kato, S.
(2007)
Vitamin K induces osteoblast differentiation through pregnane X receptor-mediated transcriptional control of the Msx2 gene.
Mol. Cell. Biol.
27
,
7947-7954
.
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Notes:
Igarashi et al. showed that expression of the Msx2 gene, which enodes a transcription factor, is induced by vitamin K treatment via a pregnane X receptor response element (PXRE) and by estrogen via an estrogen response element (ERE). Promoter analysis was performed by cloning the Msx2 promoters into the pGL3-Basic Vector, transfecting MC3T3 and ST2 cells with the pGL3-Basic constructs, treating the cell with 10nM 17β-estradiol and 10µM vitamin K, then measuring luciferase activity. The pRL-CMV Vector (2.5ng per well of a 12-well plate) was cotransfected to normalize for transfection efficiency. The ability of PXR to bind to the Msx2-PXRE was assessed by an avidin-biotin complex DNA assay. Sense and antisense oligonucleotides that were biotinylated at the 5´ end were annealed and immobilized with the TetraLink™ Tetrameric Avidin Resin. HEK293T cells were lysed with lysis buffer (10mM Tris-HCl [pH7.8], 1mM EDTA, 150mM NaCl, 0.1% NP-40) containing protease inhibitors, then centrifuged to clarify the extract. The supernatants were mixed with the DNA-TetraLink™ Resin to allow proteins to bind to the oligos, and resin was washed with lysis buffer. Bound proteins were analyzed by SDS-PAGE and Western blotting.
(0003805) |
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Products: pGL3-Basic Vector | pRL-CMV Vector | TetraLink™ Tetrameric Avidin Resin |
| 11. |
Kanayama, T., Arito, M., So, K., Hachimura, S., Inoue, J. and Sato, R.
(2007)
Interaction between sterol regulatory element-binding proteins and liver receptor homolog-1 reciprocally suppresses their transcriptional activities.
J. Biol. Chem.
282
,
10290–10298
.
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Notes:
To explore the interaction of liver receptor homolog (LRH)-1, a known suppressor of sterol regulatory element-binding protein (SREBP) transcriptional activity, human LRH-1 was reverse transcribed then amplified by PCR from total RNA from HepG2 cells. The amplification product was ligated into the pTargeT™ Mammalian Expression Vector to create pTarget-LRH1. For reporter experiments, a PCR fragment that encompassed the 1.3kb 5’-promoter region of the human small heterodimer partner (SHP) gene was cloned into the pGL3-Basic Vector (designated pSRB). The pGL3-Promoter Vector was used to construct pLRHREx3, which contains three LRH-1 response elements, and the insert was generated using synthetic oligonucleotides. HEK293 cells were cotransfected with 0.2µg of a promoter-firefly luciferase construct, 0.1µg of a SREBP expression plasmid, 10ng of phRL-TK Vector and 0.2 or 0.6µg of pTarget-LRH1. Alternatively, the cotransfected plasmids were 0.2µg of pSHP, 0.1µg of pTarget-LRH1, 10ng of phRL-TK Vector and 0.2 or 0.6µg of a SREBP expression plasmid. The pLRHREx3 construct (0.2µg) was cotransfected with 0.1µg of a LRH-1 expression plasmid, 0.2µg of pCMXPGC-1α (peroxisome proliferator activated receptor γ coactivator-1α), 10ng of phRL-TK Vector, and 0.1 or 0.3µg of a pSREBP expression vector in HEK 293 cells. Luciferase expression was assayed 48 hours post-transfection using the Dual-Luciferase® Assay Reporter System. To express SREBPs and LRH-1 in vitro, inserts were ligated into the pTNT™ Vector, synthesized using the TNT® Coupled Transcription/Translation System with radiolabeled methionine. Ten microliters of the 35S-labelled protein was then used in a GST-pulldown assay.
(0003692) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pGL3-Basic Vector | pGL3-Promoter Vector | pTNT™ Vector | TNT® SP6 Coupled Reticulocyte Lysate System | TNT® SP6 Coupled Reticulocyte Lysate System, Trial Size | TNT® T3 Coupled Reticulocyte Lysate System | TNT® T7 Coupled Reticulocyte Lysate System | TNT® T7 Coupled Reticulocyte Lysate System, Trial Size |
| 12. |
Li, Y., Zhang, Z., Robinson, G.E. and Palli, S.R.
(2007)
Identification and characterization of a juvenile hormone response element and its binding proteins.
J. Biol. Chem.
282
,
37605–37617
.
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Notes:
The authors characterized a juvenile hormone response element in Drosophila melanogaster (DmJHRE1) and identified two proteins that bound to a DmJHRE1 affinity column. Proteins eluted from the column were digested with Sequencing Grade Modified Trypsin, subjected to liquid chromatography-tandem mass spectrometry and identified as FKBP39 and Chd64. DmJHRE1 transcription regulatory activity was confirmed using reporter constructs with DmJHRE1 sequences regulating expression of firefly luciferase in Drosophila L57 and S2 cells. A vector with Renilla luciferase and the Autographa californica multicapsid nucleopolyhedrovirus IE1 promoter was used for normalization. Luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. Potential interactions between FKBP39, Chd64 and several candidates proteins for the JH receptor were examined using the MagneGST™ Pull-Down System. Each bait protein was expressed as a GST-fusion protein in E. coli and immobilized using MagneGST™ Glutathione Particles. [35S]Methionine-labeled prey proteins were expressed using the TNT® T7 Quick Coupled Transcription/Translation System.
(0003784) |
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Products: Dual-Luciferase® Reporter Assay System | MagneGST™ Pull-Down System | Sequencing Grade Modified Trypsin | TNT® T7 Quick Coupled Transcription/Translation System |
| 13. |
Kommagani, R., Payal, V. and Kadakia, M.P.
(2007)
Differential regulation of vitamin D receptor (VDR) by the p53 family: p73-dependent induction of VDR upon DNA damage.
J. Biol. Chem.
282
,
29847–29854
.
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Notes:
The authors examined transcriptional regulation of the vitamin D receptor (VDR) by p53 and p63, a member of the p53 family, under stressed and unstressed conditions. Reporter constructs with the full-length and minimal VDR promoters controlling expression of firefly luciferase were cotransfected with p53 or p63 expression constructs, and transcriptional activation of the VDR promoter was monitored using the Dual-Luciferase® Reporter 100 Assay System. Results were normalized to Renilla luciferase activity. Interaction between p73, another member of the p53 family, and the VDR promoter was examined using chromatin immunoprecipitation. The imunnopreciptated chromatin was reverse crosslinked, DNA was eluted and VDR and p21 sequences were detected by PCR using GoTaq® Green Master Mix.
(0003715) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | GoTaq® Green Master Mix |
| 14. |
Liu, Y., Kern, J.T., Walker, J.R., Johnson, J.A., Schultz, P.G., and Luesch, H.
(2007)
A genomic screen for activators of the antioxidant response element.
Proc. Natl. Acad. Sci. U S A
104
,
5205-5210
.
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Notes:
These authors screened a library of 15,000 expression cDNAs in neuroblastoma IMR-32 cells searching for genes that activate the antioxidant response element, ARE. ARE is a cis-acting enhancer element found in the 5´ flanking region of many genes that are involved in protection from oxidative stress. The library was screened using a luciferase reporter construct under the control of an ARE-containing promoter. Luminescence, indicating the presence of cDNA-activating ARE, was measured using the Bright-Glo™ Luciferase Assay System. cDNA clones showing reproducible activation were selected for further analysis. The authors tested the effect of over expression of these ARE activators on the ability to resist oxidative stress. IMR-32 cells expressing the various cDNAs were exposed to hydrogen peroxide or rotenone, and the effect on cell viability was measured using the CellTiter-Glo® Assay. Cells overexpressing the ARE-activators were more resistant to oxidative stress than controls.
(0003629) |
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Products: Bright-Glo™ Luciferase Assay System | CellTiter-Glo® Luminescent Cell Viability Assay |
| 15. |
Pandhare, J., Cooper, S.K. and Phang, J.M.
(2006)
Proline oxidase, a proapoptotic gene, is induced by troglitazone: evidence for both peroxisome proliferator-activated receptor gamma-dependent and -independent mechanisms.
J. Biol. Chem.
281
,
2044–2052
.
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Notes:
A proline oxidase (POX) antisense vector was generated by amplifying part of the POX cDNA and ligating the product into the pCI Mammalian Expression Vector in the antisense orientation. This construct was tested and validated for blocking POX mRNA expression using RT-PCR. Both PPARγ and p53 cDNAs were also cloned into the pCI Vector. The human POX promoter sequence was amplified and cloned into the NheI and HindIII sites of the pGL3-Basic Vector to create the POX-Luc reporter construct. Using several colon cancer cell lines (HT29, LoVo, HCT116, HCT15, RKO, KM12, HCC2998 and SW620), the POX-Luc construct was co-transfected with pRL-null (to normalize transfection efficiency) plus PPARγ, p53 contructs or empty vector. A PPARγ ligand was added 10 hours post-transfection and cells harvested 24–36 hours after transfection. POX promoter luciferase activity was measured using the Dual-Luciferase® Reporter Assay System and a TD-20/20 luminometer.
(0003514) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pCI-neo Mammalian Expression Vector | pGL3-Basic Vector | pRL-null Vector |
| 16. |
Cejas, P.J., Carlson, L.M., Zhang, J., Padmanabhan, S., Kolonias, D., Lindner, I., Haley, S., Boise, L.H. and Lee, K.P.
(2006)
Protein Kinase C βII plays an essential role in dendritic cell differentiation and autoregulates its own expression.
J. Biol. Chem.
280
,
28412-28423
.
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Notes:
Protein Kinase C activity was assayed in unstimulated KG1, KG1a, KG1a-neo and KG1a-PKC-βII-GFP human leukemic cells using the SignaTECT® Protein Kinase C (PKC) Assay System. For PKC-βII promoter analysis, reporter constructs were cloned into the pGL3-Basic Vector. The pRL-CMV plasmid was used as an internal control to normalize luciferase activity. Reporter assays were carried out using the Dual-Luciferase® Reporter Assay System.
(0003407) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pGL3-Basic Vector | SignaTECT® Protein Kinase C (PKC) Assay System |
| 17. |
Perez, L.E., Chandrasekar, B., Saldarriaga, O.A., Zhao, W., Arteaga, L.T., Travi, B.L. and Melby, P.C.
(2006)
Reduced nitric oxide synthase 2 (NOS2) promoter activity in the Syrian hamster renders the animal functionally deficient in NOS2 activity and unable to control an intracellular pathogen.
J. Immunol.
176
,
5519-5528
.
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Notes:
Leishmania donovani infection elicits an immune response in mice macrophages that includes the upregulation of nitric oxide synthase 2 (NOS2). Hamster and human macrophages do not exhibit an upregulation of NOS2 upon infection. The authors measured the activities of the NOS2 promoter in response to interferon-γ (IFNγ) and lipopolysaccharide treatment of mouse, hamster and human macrophages. The mouse, hamster and human NOS2 promoters were cloned into pGL3-Basic Vector and transfected into mouse macrophages by electroporation. Promoter activities were determined using the Dual Luciferase® Reporter Assay System. The pRL-null Vector was used to normalize for differences in transfection efficiency
(0003470) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pGL3-Basic Vector | pRL-null Vector |
| 18. |
de Wolf, C.J., Cupers, R.M., Bertina, R.M. and Vos, H.L.
(2006)
The constitutive expression of anticoagulant protein S is regulated through multiple binding sites for Sp1 and Sp3 transcription factors in the protein S gene promoter.
J. Biol. Chem.
281
,
17635–17643
.
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Notes:
The Protein S promoter (PROS1) fragment –5948/–1 was cloned directly 5’ to the firefly luciferase reporter gene in the pGL3-Basic Vector using the KpnI and XhoI enzyme sites. This construct, PS5948-luc, was linearized with KpnI and NdeI and subsequently subjected to progressive deletion using the Erase-a-Base® System. The size of the resulting 5’-deletion was determined by sequence analysis, and the deletion constructs were used for transient transfection assays. HepG2, HuH7, HeLa and HUVEC cells were transfected at 60–80% confluency in 12-well plates using 3µl of Tfx™-20 per microgram DNA. In each transfection, an equimolar concentration of construct was used and supplemented with an additional plasmid to keep the amount of transfected DNA constant. pRL-SV40 Vector was co-transfected as a transfection control using a 1:500 ratio to the total transfected amount of DNA in HepG2, HuH7 and HeLa cell lines, and a 1:100 ratio in transfections with HUVEC and 1 × 106 Meg01 suspension cells. Transcription factor expression vector (250ng) was co-transfected, and expression vector without the transcription factor cDNA was used as a negative control. Cell extracts were harvested at either 24 (HepG2 and HuH7) or 48 hours (Meg01, HUVEC, and HeLa) post-transfection using 250µl of Passive Lysis Buffer per well. Luciferase activity was determined using 20–100µl of lysate with the Dual-Luciferase® Reporter Assay System.
(0003510) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | Erase-a-Base® System (minus vectors & bacterial strain) | pGL3-Basic Vector | Tfx™-20 Reagent |
| 19. |
Gritsko, T., Williams, A., Turkson, J., Kaneko, S., Bowman, T., Huang, M., Nam, S., Eweis, I., Diaz, N., Sullivan, D., Yoder, S., Enkemann, S., Eschrich, S., Lee, J.H., Beam, C.A., Cheng, J., Minton, S., Muro-Cacho, C.A. and Jove, R.
(2006)
Persistent activation of stat3 signaling induces survivin gene expression and confers resistance to apoptosis in human breast cancer cells.
Clin. Cancer Res.
12
,
11–19
.
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Notes:
These authors identified survivin as a Stat3-regulated gene in breast cancer cells using microarray analysis. The authors used a pGL2 construct containing the survivin gene promoter driving firefly luciferase expression and a Promega luciferase assay system to monitor survivin promoter activity in NIH 3T3 cells. Cotransfection with vectors expressing Stat3 and v-src, which activates endogenous Stat3, increased survivin promoter activity by almost fivefold, whereas expression of a dominant-negative Stat3 variant decreased survivin promoter activity by 50%.
(0003610) |
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Products: pGL2-Basic Vector |
| 20. |
Ullah, M.S., Davies, A.J. and Halestrap, A.P.
(2006)
The plasma membrane lactate transporter MCT4, but not MCT1, is up-regulated by hypoxia through a HIF-1alpha-dependent mechanism.
J. Biol. Chem.
281
,
9030-9037
.
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Notes:
Monocarboxylate transporters (MCT) transport lactic acid across the cell membrane. The promoters of 4 MCT family members (MCT1, MCT2, MCT3 and MCT4), were amplified by PCR and cloned into the pGEM®-T Easy Vector. The sequences were confirmed, and the promoters were cloned into the pGL3-Basic Vector. The Dual-Glo™ Luciferase Assay System was used to quantitate promoter activity under basal and hypoxic conditions in HeLa cells. The pRL-SV40 Vector was used to normalize for differences in transfection efficiency.
(0003463) |
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Products: Dual-Glo® Luciferase Assay System | pGEM®-T Easy Vector System I | pGEM®-T Easy Vector System II | pGL3-Basic Vector | pRL-SV40 Vector |
| 21. |
Lee, S.J., Jang, B.C., Lee, S.W., Yang, Y.I., Suh, S.I., Park, Y.M., Oh, S., Shin, J.G., Yao, S., Chen, L. and Choi, I.H.
(2006)
Interferon regulatory factor-1 is prerequisite to the constitutive expression and IFN-gamma-induced upregulation of B7-H1 (CD274).
FEBS Lett.
580
,
755-762
.
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Notes:
Many cancer cells upregulate the co-signaling molecule B7-H1, confering resistance to anti-tumor immunity. The ability of interferon regulatory factor-1 (IRF-1) to upregulate B7-H1 expression was characterized by cloning fragments of the B7-H1 promoter upstream of the firefly luciferase reporter gene in the pGL3-Basic Vector and monitoring luciferase expression using the Dual Luciferase® Reporter Assay System. Firefly luciferase measurements were normalized using Renilla luciferase (pRL-CMV Vector). Putative IRF-1 binding sites in the B7-H1 promoter were identified using the Gel Shift Assay System. RT-PCR was used to examine B7-H1 mRNA levels in interferon-γ-treated or untreated A549 cells exposed to various concentrations of IRF-1 siRNA. cDNA synthesis was performed with the ImProm-II™ Reverse Transcription System.
(0003451) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | Gel Shift Assay Core System | Gel Shift Assay System | ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System | pGL3-Basic Vector | pRL-CMV Vector |
| 22. |
Udayakumar, T.S., Belakavadi, M., Choi, K.H., Pandey, P.K. and Fondell, J.D.
(2006)
Regulation of Aurora-A kinase gene expression via GABP recruitment of TRAP220/MED1.
J. Biol. Chem.
281
,
14691–14699
.
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Notes:
TRAP220/MED1 is amplified in estrogen receptor-positive breast cancer cells and has been shown to interact with a number of transcription factors essential for cell growth and development including BRCA-1 and p53. TRAP220/MED1 is a subunit of the TRAP/Mediator coactivator complex. These authors used RNA interference to reduce TRAP220/MED1 expression by >90%, then microarray analysis to identify genes that were downregulated after TRAP220/MED1 depletion. One such gene was Aurora-A serine/threonine kinase. The authors created Aurora-A-firefly luciferase constructs to determine the effect of TRAP220/MED1 depletion on Aurora-A promoter activity. As a positive control, the authors used a thyroid hormone (T3)-responsive firefly luciferase construct to show that depletion of TRAP220/MED1, which is known to play a role in nuclear receptor-mediated gene activation, interferes with thyroid hormone receptor-mediated activation of T3-responsive genes. Luciferase reporter gene activity was measured using the Dual Luciferase Reporter Assay System, and results were normalized to Renilla luciferase expression from the pRL-TK Vector.
(0003607) |
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Products: Dual-Luciferase® Reporter Assay System | pRL-TK Vector |
| 23. |
Baldwin, A., Huh, K-W. and Mϋnger, K.
(2006)
Human papillomavirus e7 oncoprotein dysregulates steroid receptor coactivator 1 localization and function.
J. Virol.
80
,
6669-6677
.
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Notes:
The MagneGST™ Protein Purification System was used to purify GST fusion proteins of the oncoprotein HPV16 E7 or various mutants of HPV16 E7. The purified GST fusion proteins were used for in vitro binding experiments with steroid receptor coactivator 1 (SRC-1), which was produced using the TNT® T7 Coupled Wheat Germ Extract System and labeled with the Transcend™ Non-Radioactive Translation Detection System. GST pull-down assays were resolved by Western analysis using streptavidn-horseradish peroxidase and alpha-GST. To determine the effects of endogenously expressed HPV16 E7 on SCR-1-mediated transcription, luciferase reporters under the control of either the IL-8 promoter or an artificial promoter containing three estrogen response elements repeats (3 × EREs) were cotransfected with a Renilla control vector into two human cervical cancer lines (C33A and CaSki) using either the Transfast™ Transfection Reagent or another commercial transfection reagent. The Dual-Luciferase® Reporter Assay was then used to determine luciferase activity to functionally map the E7-interacting domain and to determine the effects of high- and low-risk PHV E7s on SRC-1-mediated transcription.
(0003459) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | MagneGST™ Glutathione Particles | MagneGST™ Protein Purification System | TNT® T7 Coupled Wheat Germ Extract System | Transcend™ Chemiluminescent Non-Radioactive Translation Detection System | Transcend™ Colorimetric Non-Radioactive Translation Detection System | Transcend™ tRNA | TransFast™ Transfection Reagent |
| 24. |
Böhm, C., Seibel, N., Henkel, B., Steiner, H., Haas, C. and Hampe. W.
(2006)
SorLA signaling by regulated intramembrane proteolysis.
J. Biol. Chem.
281
,
14547–14553
.
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Notes:
To test the ability of SorICD to stimulate trascription of a Gal4-dependent luciferase promoter, COS-7 cells were transfected with pG5luc, pBIND, or pBINDSorICD from the CheckMate™ Mammalian Two-Hybrid System. Luciferase expression was analyzed using the Bright-Glo™ Luciferase Assay System.
(0003376) |
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Products: Bright-Glo™ Luciferase Assay System | CheckMate™ Mammalian Two-Hybrid System |
| 25. |
Talmud, P.J., Palmen, J., Putt, W., Lins, L., and Humphries, S.E.
(2005)
Determination of the functionality of common APOA5 polymorphisms.
J. Biol. Chem.
280
,
28215-28220
.
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Notes:
The authors investigated common variants of the APOA5 gene that have been associated with differences in plasma triglyceride (TG) levels. PCR fragments containing either the –1131T --> C promoter variant or containing both the –1131T --> C and –3G --> A promoter variants were cloned into the pGEM®-T Vector System. The fragments were subsequently cloned into the pGL3 Basic Vector and transiently transfected into Huh7 and HepG2 cells along with the luciferase control vector, pRL-TK. The cells were lysed 48 hours after transfection and Luciferase activity was measured with the Dual-Luciferase® Reporter Assay System. The function of the 1891T --> C variant in the 3´ UTR was tested the same way; with the exception that site-directed mutagenesis was performed to introduce the T --> C at position 1891 before the fragment was cloned into the pGL3 Basic Vector. The functionality of the Kozak sequence –3A --> G variant was determined by cloning cDNAs into the pGEM®-7Zf Vector. Transcription/translation experiments were performed using the TNT® Quick Coupled Transcription/Translation System and the proteins were labeled using the FluorTect™ GreenLys System. In addition, a primer extension inhibition assay was performed using capped mRNAs generated with the Riboprobe® System –T7 and the Ribo m7G Cap Analog. Ribosome binding reactions were performed using the Rabbit Reticulocyte Lysate System, Nuclease Treated.
(0003460) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | FluoroTect™ GreenLys in vitro Translation Labeling System | pGEM®-7Zf(–) Vector | pGEM®-7Zf(+) Vector | pGEM®-T Vector System I | pGEM®-T Vector System II | pGL3-Basic Vector | Rabbit Reticulocyte Lysate System, Nuclease Treated | Ribo m7G Cap Analog | Riboprobe® System—T7 | TNT® T7 Quick Coupled Transcription/Translation System | TNT® T7 Quick Coupled Transcription/Translation System, Trial Size |