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Mol. Endocrinol. 23(10), 1587-602. Insulin acts through FOXO3a to activate transcription of plasminogen activator inhibitor type 1. 2011

Jag, U.R., Zavadil, J. and Stanley, F.M.

Notes: The authors used the Beta-Glo® Assay System to determine the transfection efficiency of electroporation. Control of transfection efficiency was done by using a rous sarcoma virus-β-galactosidase expression plasmid at 0.5μg/electroporation. The Plasminogen activator inhibitor-1 (PAI-1) was expressed with a luciferase reporter in a varitey of cell lines to show that the effect of insulin to increase PAI-1 is increased by expression of Forkhead-related transcription factors (Fox). To identify which Fox mediated the effects of insulin on PAI expression, small interfering RNAs specific for each Fox were tested along with chromatin cross-linking and ChIP experiments. The authors were able to demonstrate that insulin acts through FoxO3a to increase PAI-1 gene expression, a major regulator of fibrinolysis. (4128)

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Expert Opin. Drug Metab. Toxicol. 4, 103–120. Bioluminescent assays for ADMET 2008

Cali, J.J., Niles, A., Valley, M.P., O’Brien, M.A., Riss, T.L., and Shultz, J.

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. (3926)

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Appl. Environ. Microbiol. 74, 3667-3671. Rapid screening of quorum-sensing signal N-acyl homoserine lactones by an in vitro cell-free assay. 2008

Kawaguchi, T., Chen, Y.P., Norman, R.S. and Deecho, A.W.

Notes: The authors of this paper describe the development of a cell-free assay system derived from Agrobacterium tumefaciens NTL4(pCF218) (pCF372), which expresses β-galactosidase under the control of a tra1 promoter. The tra1 promoter is responsive to quorum-sensing signals. The assay was developed to address time-consuming cell conditioning and incubation times of the standard whole-cell assays used to detect quorum-sensing signals in natural systems. Replacing the X-Gal substrate with the luminescent Beta-Glo® substrate increased the assay sensitivity by tenfold. (3936)

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Cancer Res. 67, 1239–1245. TSP50 encodes a testis-specific protease and is negatively regulated by p53. 2007

Xu, H., Shan, J., Jurukovski, V., Yuan, L., Li, J. and Tian, K.

Notes: TSP50 is a testis-specific gene found to be overexpressed in human breast cancer tissue. Of interest is a putative p53 binding site in the TSP50 promoter. To examine what effect p53 may have on TSP50 expression, the TSP50 promoter was cloned into a pGL3 Luciferase Reporter Vector and cotransfected with a wildtype or R249S mutant p53 and a control vector, pCMV/β-galactosidase, into HeLa, HEK293 and paired MCF7 cells. After 24 hours, the cells were assessed for luciferase expression using the Luciferase Assay System and normalized to β-galactosidase expression, which was measured using the Beta-Glo® Assay System. (3598)

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RNA 11, 447–458. Binding specificity and mRNA targets of a C. elegans PUF protein, FBF-1. 2006

Bernstein, D., Hook, B., Hajarnavis, A., Opperman, L. and Wickens, M.

Notes: PUF proteins act to reduce expression upon binding to the 3´UTR of target mRNA sequences. In this study, the binding of various PUF proteins (including C. elegans FBF-1) to known PUF binding sites was evaluated using a yeast three-hybrid system. For this assay, various PUF proteins fused to the Gal4 activation domain were expressed in pACT and pACT2 plasmids, and DNA oligos expressing PUF binding RNA sequences were cloned into the plasmid pIIIA/MS2-2. Assays were then performed in the yeast strain YBZ-1. β-galactosidase expression, indicating interaction between each PUF protein and the various binding sites, was measured using both solid- (colony lift) and liquid-phase assays. For the liquid-phase assays, β-galactosidase expression was measured using the Beta-Glo® Assay System. Cells were grown in selective media to an O.D.600 of 0.1–0.3, then mixed with an equal volume of Beta-Glo® Assay Reagent. After one hour, luminescence was measured. Luminescence values were normalized to cell number. The results of these binding assays were used to formulate a consensus PUF binding sequence, which was then used to screen a C. elegans 3´ UTR database for potential FBF-1 target sequences. (3458)

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Cancer Res. 65, 10024–10031. Epigenetic regulation of WTH3 in primary and cultured drug-resistand breast cancer cells. 2005

Tian, K., Jurukovski, V., Wang, X-P., Kaplan, M.H. and Xu, H.

Notes: Methylation of the WTH3 promoter region has been associated with multidrug resistance in breast cancer cells in vitro. In this study, the effect of WTH3 in primary cultured multidrug resistant cells was evaluated, and the influence of a frequently methylated CpG23 region on WTH3 promoter activity was investigated using a luciferase reporter assay system. Promoter regions containing wildtype or mutated CpG regions were cloned into the pGL3 Vector and transfected into paired MCF7 cell lines, along with a control pGL3 Vector without insert, and a plasmid containing the β-galactosidase gene as a transfection efficiency control. Luciferase activity was measured using the Steady-Glo® Luciferase Assay System, and β-galactosidase activity was determined using the Beta-Glo® Assay System. Luciferase activities of the wildtype and mutant promoter regions were compared after normalization to β-galactosidase activity and protein concentration. (3457)

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Anal. Biochem. 336, 102–107. Homogeneous enzyme immunoassay modified for application to luminescence-based biosensors. 2005

Yang, X., Janatova, J. and Andrade, J.D.

Notes: CEDIA assays (cloned enzyme donor immunoassays) utilize antibodies to specific analytes that can also bind one of the two beta-galactosidase fragments. The beta-galactosidase fragments can associate with each other provided that the fragment bound to the analyte is not immunoabsorbed, thus blocking the fragment interaction.  The Beta-Glo® Assay System was compared to colorimetric and chemiluminescent detection systems for use in a CEDIA assay with valporic acid as analyte. The Beta-Glo® Assay System gave the best performance in this assay system.  (3224)

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RNA 11, 227-233. RNA-protein interactions in the yeast three-hybrid system: Affinity, sensitivity, and enhanced library screening. 2005

Hook, B., Bernstein, D., Zhang, B., and Wickens, M.

Notes: These authors describe use of the Beta-Glo® Assay System to analyze beta-galactosidase reporter enzyme concentrations from yeast three-hybrid experiments. The authors optimized the Beta-Glo® assay with various Saccharomyces cerevisiae strains. The optimal incubation time using the Beta-Glo® reagent on yeast was one hour, whether the cells were in stationary or exponential growth phase. Data is presented as arbitrary light units from the luminometer. (3295)

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J. Biol. Chem. 279, 29066–29074. BCL-2 translation is mediated via internal ribosome entry during cell stress. 2004

Sherrill, K.W., Byrd, M.P., Van Eden, M.E. and Lloyd, R.E.

Notes: In this paper, the effect of a 5’ untranslated region from the Bcl-2 gene transcripts on firefly and Renilla reporter constructs was evaluated. A number of studies were performed using various single- and dual-reporter constructs containing the Bcl-2 5’ UTR. These constructs were transfected into 293T cells and assayed for luciferase activity using the Dual-Luciferase® Reporter Assay System. Transfection studies with firefly luciferase mRNA constructs were also performed. In these experiments, firefly luciferase levels were measured using the Luciferase Assay System.  Transfections were normalized using the pSV-β-Galactosidase Control Vector and the Beta-Glo® Assay System.  (3125)

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Clin. Can. Res. 10, 3169-3178. Suppression of constitutive and tumor necrosis factor alpha-induced nuclear factor (NF)-kappaB activation and induction of apoptosis by apigenin in human prostate carcinoma PC-3 cells: correlation with down-regulation of NF-kappaB-responsive genes. 2004

Shukla, S. and Gupta, S.

Notes: The Beta-Glo® Assay System was used to normalize luciferase readings in dual-reporter transfection studies.  In these experiments, human prostate carcinoma PC-3 cells were transfected with a NF-κB factor-driven luciferase reporter construct and a undefined beta-galactosidase reporter construct.  Cells were treated with 20 and 40µM apigenin for 16 h or with 10ng/ml TNF-α for 30 minutes.  Luciferase reporter enzyme expression was assessed with the Luciferase Assay System.  (3054)

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