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Nucl. Acids Res. 37, 78–95. Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage. 2009

Wilson, P.M., Fazzone, W., LaBonte, M.J., Lenz, H.J. and Ladner, R.D.

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. (4031)

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FEBS Lett. 580, 755-762. Interferon regulatory factor-1 is prerequisite to the constitutive expression and IFN-gamma-induced upregulation of B7-H1 (CD274). 2006

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.

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. (3451)

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Drug Metab. Dispos. 33, 1244–53. Sp1 and Sp3 regulate basal transcription of the human CYP2F1 gene. 2005

Wan, J., Carr, B.A., Culter, N.S., Lanza, D.L., Hines, R.N. and Yost, G.S.

Notes: The human lung cell line A549 and the human liver cell line were transiently transfected with 0.1µg of CYP2F1 reporter constructs and 0.001µg of pRL-TK Vector using FuGENE® 6 Reagent in 96-well plates. For cotransfection studies, cells were transfected with 0.1µg of the reporter construct, 0.002µg of pRL-TK plasmid, and 0.5 or 0.2µg of Sp1, Sp3 or empty expression vectors, with the total transfected DNA remaining at 0.2µg. Reporter activity was assessed using the Dual-Luciferase® Reporter Assay System.

SL-2 cells were seeded in 24-well plates and cotransfected with 0.3µg of CYP2F1 reporter plasmid and 0.3µg of pPac/Sp1, pPac/Sp2 or empty expression vector. The total amount of plasmid DNA used for each transfection was 0.9µg. The DNA and FuGENE® 6 were added at a 3:1 ratio. Activities were assessed using the Dual- Luciferase® Reporter Assay System.

The Gel Shift Assay System was used to identify Sp1-like sites in the promoter of the human CYP2F1 using EMSA (electromobility shift analysis). (4269)

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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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J. Am. Soc. Nephrol. 11, 1607-19. The decorin high glucose response element and mechanism of its activation in human mesangial cells. 2000

Wahab, N.A., Parker, S., Sraer, J.D., and Mason, R.M.

Notes: Hyperglycemia seems to be involved in the pathogenesis of diabetic nephropathy. The authors identify regulatory promoter elements involved in gene regulation under hyperglycemic conditions and characterize some of the signaling pathways involved. Immunocytochemistry of human mesangial cells grown under normal and high glucose conditions was performed with the Anti-ACTIVE® MAPK pAb to localize dually phosphorylated, active MAPK protein. Cells were fixed in 3.7% paraformaldehyde, permeabilized with 0.1% Triton® X-100 and stained with the Anti-ACTIVE® MAPK pAb overnight at 4°C, or for 1 hour at 37°C. Gel shift assays were performed to identify a regulatory element which appears to be responsive to glucose concentration. Gel shift assays were carried out using Promega's Gel Shift Assay System. (2394)

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Proc. Natl. Acad. Sci. USA 95, 5795-5800. Amyloid beta-peptide stimulates nitric oxide production in astrocytes through an NFκB-dependent mechanism. 1998

Akama, K.T., Albanese, C., Pestell, R.G., and Van Eldik, L.J.

Notes: Astrocytes were transfected using Tfx™-50 Transfection Reagent at a 1:1 DNA/reagent ratio (optimal). Cells were exposed to the DNA:lipid complex for 1-2 hours. (2213)

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J. Immunol. 161, 430-439. Angiotensin II participates in mononuclear cell recruitment in experimental immune complex nephritis through nuclear factor-kappa B activation and monocyte chemoattractant protein-1 synthesis. 1998

Ruiz Ortega, M., Bustos, C., Hernandez Presa, M.A., Lorenzo, O., Plaza, J.J., Egido, J.

Notes: Promega's Gel Shift Assay System was used to perform EMSAs on cell lysates using NF-kappaB oligonucleotide probe. HeLa Nuclear Extracts were used as a positive control for NF-kappaB binding activity. (0465)

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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. Biol. Chem. 273, 13097-13103. Sp1 cooperates with the ets transcription factor, GABP, to activate the CD18 (beta2 leukocyte integrin) promoter 1998

Rosmarin, A.G., Luo, M., Caprio, D.G., Shang, J., Simkevich, C.P.

Notes: The Recombinant Human SP1 protein was used for gel shift assay at 1 footprint unit per assay. As directed in the Gel Shift Assay System Technical Bulletin, BSA was added to the reaction. The protein was also supershifted with an anti-SP1 antibody. (0457)

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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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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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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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Cell 67, 1075-1087. B cell lymphoma-associated chromosomal translocation involves candidate oncogene lyt-10, homologous to NF-kappa B p50. 1991

Neri, A., Chang, C.C., Lombardi, L., Salina, M., Corradini, P., Maiolo, A.T., Chaganti, R.S., Dalla Favera, R.

Notes: The Wheat Germ Extract was used to generate proteins which, in combination with the Gel Shift Assay System, were used for gel shift assays. (0651)

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