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Citations Search

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Sci. Rep. 8(1), 638. Effective photo-enhancement of cellular activity of fluorophore-octaarginine antisense PNA conjugates correlates with singlet oxygen formation, endosomal escape and chromophore lipophilicity. 2018

Yarani R., Shiraishi T., Nielsen P.E.

Notes: Antisense fluorophore octaarginine peptide nucleic acid (PNA) conjugates are investigated cellular activity using a photochemical internalization (PCI) drug delivery method. PCI utilizes light to generate reactive oxygen species that can damage the endosomal membrane and lead to drug release. Specifically, cellular localization and antisense activity was monitored for two conjugates, tetramethylrhodamine and Alexa Fluor 555. Luminescence and cell viability were measured using the Bright-Glo™ Assay and CellTiter-Glo® Assay, respectively. (5172)

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PLoS Pathogens 14, e1007057. Plasmodium parasite exploits host aquaporin-3 during liver stage malaria infection. 2018

Posfai, D., Sylvester, K., Reddy, A., Ganley, J.G., Wirth, J., Cullen, Q.E., Dave, T., Kato, N., Dave, S.S., and Derbyshire, E.R.

Notes: Researchers studied the liver stage of the Plasmodium parasite (which causes malaria), including RNA sequencing, parasite load and cell viability. (5035)

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Cell Death Dis. 9(2), 41. Sensitizing tumor cells to conventional drugs: HSP70 chaperone inhibitors, their selection and application in cancer models. 2018

Lazarev V.F., Sverchinsky D.V., Mikhaylova E.R., Semenyuk P.I., Komarova E.Y., Niskanen S.A., Nikotina A.D., Burakov A.V., Kartsev V.G., Guzhova I.V., Margulis B.A.

Notes: Heat shock protein 70 (Hsp70) inhibitors have shown substantial anticancer activity, however many are highly cytotoxic towards non-cancerous cells. This paper developed new drug discovery assays for compounds that reduce the chaperone activity of Hsp70. Chaperone and refolding activity were monitored using the Bright-Glo™ Luciferase Assay System. For the refolding assay, cells were heat shocked leading to luciferase denaturation. Active Hsp70 chaperone activity lead to refolding and luciferase signal. A novel compound, AEAC, showed two-fold lower cytotoxicity, as measured by the CytoTox 96® Non-Radioactive Cytotoxicity Assay, and inhibition of Hsp70 activity. (5173)

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Proc. Natl. Acad. Sci. USA 108, 18488–93. Discovery of β-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy. 2011

Allen, J.A., Yost, J.M., Setola, V., Chen, X., Sassano, M.F., Chen, M., Peterson, S., Yadav, P.N., Huang, X.P., Feng, B., Jensen, N.H., Che, X., Bai, X., Frye, S.V., Wetsel, W.C., Caron, M.G., Javitch, J.A., Roth, B.L. and Jin, J.

Notes: This paper explored potential compounds as agonists of dopamine D2 receptor (D2R) with a bias toward β-arrestin signaling. Based on the aripiprazole scaffold, compounds were synthesized and tested in a D2-mediated Gi-coupled isoproterenol-stimulated cAMP production assay using HEK293T cells expressing D2R transfected with pGloSensor™-22F cAMP Plasmid. Assessing β-arrestin recruitment to agonist-stimulated receptors was determined using HTLA cells stably expressing β-arrestin-TEV protease and a tetracycline transactivator-driven luciferase exposed to agonist or D2 test ligand with or without reference agonist. After 18 hours, medium was removed from the cells, 1X Bright-Glo™ Reagent added and luminescence measured. (4518)

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Mol. Pharmacol. 80, 367–77. Quantifying Ligand Bias at Seven-Transmembrane Receptors 2011

Rajagopal, S., Ahn, S., Rominger, D.H., Gowen-MacDonald, W., Lam, C.M., DeWire, S.M., Violin, J.D., and Lefkowitz, R.J.

Notes: The authors used stably transfected HEK cells expressing the human β2 adrenergic receptor, (β2AR) where the C-terminal tail was replaced with the C-terminal tail of the V2 vasopressin receptor (to increase signal-to-noise ratio) followed by a Tobacco Etch Virus (TEV) protease cleavage site and a tetracycline-controlled transcription factor (tTA). A second construct encoding β-arrestin 2 fused to TEV protease was also transfected into the same cell line. To follow the recruitment of β–arrestin upon ligand stimulation of the β2AR, the authors detected the cleavage of tTA and its translocation to the nucleus where it transcribed a stably expressing luciferase reporter gene. The Bright-Glo™ Luciferase Assay Reagent was used to detect luciferase activity and confirm positive recruitment of β-arrestin. The luminescent GloSensor™ cAMP Assay was used to assess ligand stimulation of the β2AR signaling through G proteins, resulting in an increase in cAMP. The authors used both of these assays to quantify ligand bias and identify weakly biased compounds of the β2AR and angiotensin II type 1A receptors. A number of known compounds were assessed to show the value of this strategy in helping to decipher complex signaling pathways. This approach may be useful in the development of novel biased ligands for therapeutic use. (4146)

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Proc. Natl. Acad. Sci. USA 106, 2412–7. Identification of compounds that potentiate CREB signaling as possible enhancers of long-term memory. 2009

Xia, M., Huang, R., Guo, V., Southall, N., Cho, M.H., Inglese, J., Austin, C.P. and Nirenberg, M.

Notes: In this study, small molecule enhancers of cAMP response element binding (CREB) were studied using quantitative high-throughput screening. After an initial screen of 73,000 compounds, 1,800 compounds were classified as potentiators of CREB activity. A second screening to confirm the compound potential was performed using the GloResponse™ CRE-luc2P HEK293 Cell Line. Five microliters of cells in assay medium were seeded in 1,536-well plates at a density of 2,500 cells/well. The next day, 23 nl of compound in DMSO or DMSO alone was dispensed into each well, then 1 μl of NKH477 (final concentration, 200 nM) or media alone was added to the assay plates. After incubating the cells for 4 hours at 37 °C, 6 μl of Bright-Glo™ Luciferase Assay Reagent was added to each well, incubated at room temperature for 10 minutes and the luminescence measured. (4004)

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J. Biomol. Scr. 13, 591-608. High-throughput screening-based identification of paramyxovirus inhibitors. 2008

Yoon, J.-J., Chawla, D., Pall, T., Ndungu, M., Du, Y., Kurtkaya, S., Sun, A., Snyder, J.P. and Plemper, R.K.

Notes: The authors describe an HTS assay to screen for inhibitors of measles virus infection. The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess toxicity of all confirmed hit compounds from the primary screen. A luminescent assay was used to assess cell-to-cell fusion in the presence candidate compounds that appeared to inhibit entry. Effector cells that expressed the T7 polymerase and measles virus H and F envelope proteins were overlaid on target cells expressing firefly luciferase under the control of a T7 polymerase promoter. Inhibition of fusion should reduce luciferase expression compared to a positive fusion control. Ten of the eleven compounds tested caused a dose-dependent reduction in luciferase expression, suggesting they block viral entry into cells. Luminescence was detected using the Bright-Glo™ Assay System. (3933)

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J. Mol. Biol. 381, 956–74. Involvement of the second extracellular loop and transmembrane residues of CCR5 in inhibitor binding and HIV-1 fusion: Insights into the mechanism of allosteric inhibition. 2008

Maeda, K., Das, D., Yin, P.D., Tsuchiya, K., Ogata-Aoki, H., Nakata, H., Norman, R.B., Hackney, L.A., Takaoka, Y. and Mitsuya, H.

Notes: The authors examined the role of specific amino acids within the second extracellular loop (ECL2) of C-C chemokine receptor 5 (CCR5), which is a coreceptor for human immunodeficiency virus type 1, in HIV-1-mediated cell fusion. The authors substituted single and multiple amino acids in ECL2 by site-directed mutagenesis, transfected MAGI cells with these CCR5 mutations, then monitored the effect of the mutations on the magnitude of cell-cell fusion. Their cell fusion assay used the pLTR-LucE plasmid, which encodes firefly luciferase and is transcriptionally activated by the HIV-1 tat protein. When tat+ cells fuse with pLTR-LucE+ cells, transcription of the luciferase gene is activated, and the level of luminescence becomes a measure of cell fusion. To perform the cell fusion assay, the researchers combined tat+, env+ 293T cells and pLTR-LucE+, CCR5+ MAGI cells for 6 hours, then measured luciferase activity using the Bright-Glo™ Luciferase Assay System. This assay allowed the researchers to identify amino acids within ECL2 that are involved in HIV-1-mediated cell fusion. (3971)

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Proc. Natl. Acad. Sci. USA 105, 4364-4369. Production and sequence validation of a complete full length ORF collection for the pathogenic bacterium Vibrio cholerae. 2008

Rolfs, A., Montor, W.R., Yoon, S.S., Hu, Y., Bhullar, B., Kelley, F., McCarron, S., Jepson, D.A., Shen, B., Taycher, E., Mohr, S.E., Zuo, D., Williamson, J,. Mekalanos, J. and Labaer, J.

Notes: These authors prepared a complete collection of Vibrio cholerae ORF clones using an automated amplification and cloning procedure. They then tested this set of clones for protein expression and capture using a nucleic acid programmable protein array method. To do this, they used the TNT® T7 Coupled Reticulocyte Lysate System to perform in situ transcription/translation of cDNA clones containing a GST fusion tag. Proteins were captured using an anti-GST antibody and subjected to further analysis. (3879)

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Proc. Natl. Acad. Sci. USA 104, 5205-5210. A genomic screen for activators of the antioxidant response element. 2007

Liu, Y., Kern, J.T., Walker, J.R., Johnson, J.A., Schultz, P.G., and Luesch, H.

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. (3629)

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Cancer Res. 67, 1254-1261. A small-molecule enhancer of signal transducer and activator of transcription 1 transcriptional activity accentuates the antiproliferative effects of IFN-γ in human cancer cells 2007

Lynch, R.A., Etchin, J., Battle, T.E. and Frank, D.A.

Notes: STAT1 is a transcription factor that is involved in a variety of cellular processes and behaves like a tumor suppressor in many ways. It is associated with apoptosis, inhibition of cyclin-dependent kinases, and it may mediate the antitumor effects of IFN-γ. The authors of this study designed a bioluminescent reporter assay to identify small molecules that enhance STAT1-dependent gene expression. The bioluminescent assay was performed in 384-well plates using both stably and transiently transfected cell lines. The screening assay was robust, producing a Z´factor value of 0.92, and it identified three compounds that specifically enhanced STAT1-dependent transcriptional activity. Firefly luciferase activity in stable cell lines was assessed using the Bright-Glo® Luciferase Assay System; luciferase activity in the transiently transfected cells was monitored using the Dual-Luciferase® Assay System. Viability of cells in culture was monitored using the CellTiter-Glo® Assay. (3732)

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Assay Drug Dev. Technol. 5, 127–136. Bioluminescent assays for high-throughput screening 2007

Fan, F. and Wood, K.V.

Notes: The authors of this paper review bioluminescent assay technologies, discussing HTS reporter, cell-based and luciferase biosensor assays. They divide luminescent assays into three basic categories: assays that measure ATP concentration (cell viability and kinase assays), assays that measure changes in luciferase levels (reporter assays, GPCR assays), and assays that measure changes in luciferin levels (protease [including caspase], P450 and MAO assays). (3737)

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Proc. Natl. Acad. Sci. USA 104, 270-275. Identification of NVP-TAE684, a potent, selective and efficacious inhibitor of NPM-ALK 2007

Galkin, A.V., Melnick, J.S., Kim, S., Hood, T.L., Li, N., Li, L., Xia, G., Steensma, R., Chopiuk, G., Jiang, J., Wan, Y., Ding, P., Liu, Y., Sun, F., Schultz, P.G., Gray, N.S. and Warmuth, M.

Notes: NVP-TAE684 was identified as an inhibitor of the constitutive anaplastic lymphoma kinase (ALK) activity associated with the NPM-ALK fusion. NPM-ALK fusion protein is created by translocation event characteristic of anaplastic large-cell lymophomas. NVP-TAE684 was screened against a panel of 35 Ba/F3 cell lines expressing a variety of tyrosine kinases constitutively activated by fusion to TEL. The CellTiter-Glo® Luminescent Cell Viability Assay was used to detect any decrease in viability as a result of treatment with NVP-TAE684. The inhibitory effect of NVP-TAE684 was specific for ALK-associated cell proliferation. In a secondary screen to determine potency, TAE684 was screened against two human anaplastic large-cell lymphoma cell lines. Inhibition of proliferation correlated with dosage and was assessed using the Bright-Glo® Luciferase Assay System. (3738)

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J. Biomol. Scr. 12, 546–559. Optimization procedure for small interfering RNA transfection in a 384-well format. 2007

Borawski, J., Lindeman, A., Buxton, F., Labow, M. and Gaither, L.A.

Notes: A lentiviral expression vector containing the firefly luciferase gene from a pGL3 Vector was transduced into SKOV3 cells in 384-well plates, transfected with various siRNAs and analyzed 72 hours later. The luciferase expression was determined using the Bright-Glo™ Luciferase Assay System and cell viability assessed using the CellTiter-Glo® Luminescent Cell Viability Assay. (3729)

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J. Biol. Chem. 281, 14547–14553. SorLA signaling by regulated intramembrane proteolysis. 2006

Böhm, C., Seibel, N., Henkel, B., Steiner, H., Haas, C. and Hampe. W.

Notes: To test the ability of SorICD to stimulate transcription 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. (3376)

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J. Biomol. Scr. 10, 1-12. A high-throughput screen to identify inhibitors of amyloid beta-protein precursor processing 2005

Bakshi, P., Liao, Y-F., Gao, J., Ni, J., Stein, R., Yeh, L-A., Wolfe, M.S.

Notes: A key component in the pathogenesis of Alzheimer's disease is cerebral accumulation of amyloid-beta protein (Aβ). Aβ is produced by proteolysis of amyloid-β-protein precursor (APP) by ß- and gamma-secretases, thus these enzymes are considered important drug targets for Alzheimer's disease. Existing assays for assessing inhibition of alpha-, beta- and gamma-secretases include HPLC or ELISA assays that are cumbersome, expensive and not well-suited to high-throughput screening. The authors developed a luciferase reporter system to identify new molecules that inhibit APP processing. They then successfully interfaced this sensitive, specific and quantitative assay with a high-throughput screen, useful for identifying both inhibitors and stimulators of APP processing. (3775)

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J. Biol. Chem. 278 (52), 52739-52746. Expression of a human surfactant protein C mutation associated with interstitial lung disease disrupts lung development in transgenic mice.  2005

Bridges, J.P., Wert, S.E., Nogee, L.M. and Weaver, T.E.

Notes: A minimal promoter from the gene encoding BiP was cloned into the pGL3-Basic Vector. The resulting construct was transfected into HEK293 cells and, 48 hours post transfection, cell lysates were prepared using Glo Lysis Buffer. The Bright-Glo™ Luciferase Assay System was then used to assess levels of luciferase expression.  As a transfection control, cells were co-transfected with the pSV-β-Galactosidase Control Vector.  (3229)

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J. Biomol. Scr. 10, 456-462. Homogeneous high-throughput screening assays for HIV-1 integrase 3' processing and strand transfer activities 2005

Wang, Y., Klock,H., Yin, H., Wolff, K., Bieza, K., Niswonger, K., Matzen, J., Gunderson, D., Hale, J, Lesley, S., Kuhen, K., Caldwell, J, Brinker, A.

Notes: HIV-1 integrase (HIV-IN) catalyzes a two-step reaction that results in HIV-1 provirus incorporation into the host cell genome. The steps involve an endonucleolytic 3’-processing (3P) followed by a strand transfer (ST) reaction. In past research it has been demonstrated that small molecule inhibitors of the in vitro activity of the 3P reaction only, lack antiviral activity, while inhibition of the HIV-IN ST reaction has been shown to be the key to effective suppression of viral replication in vivo. In the ST inhibitor realm, two series of diketo acids and naphthridine carboxamides have demonstrated antiviral activity in cell culture-based models. However, no compounds have completed clinical trials so far, due to either limited potency or high toxicity. Traditional integrase assays have been low-throughput, gel-based assays involving radiolabeled oligonucleotides. More recently high-throughput assays have been developed, but these microtiter-based assays, while amenable to automation, are complicated and labor intensive due to the need for plate coating and washing. The authors describe development of two robust, homogeneous time-resolved fluorescent energy transfer (TR-FRET)-based assays for HIV-IN 3P and ST reactions that are optimized for 384-well amd 1536-well plate formats. A screen for HIV-IN inhibitors was performed on a 1.36 x 106 compound library, resulting in a series of novel HIV-IN inhibitors that preferentially block integrase ST activity and show potential for further development as new antiviral drugs. (3776)

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J. Biol. Chem. 278 (22), 19691-19701. Complex transcription and splicing of odorant receptor genes. 2003

Volz, A., Ehlers, A., Younger, R., Forbes, S., Trowsdale, J., Schnorr, D., Beck, S. and Ziegler, A.

Notes: Total RNA was isolated from human testes using the RNAgents® Total RNA Isolation System. The isolated RNA was used in reverse transcription reactions to make cDNAs of HLA-linked OR genes. Promoter regions of HLA-linked OR genes were cloned into the pGL3-Basic Vector. The constructs were then transfected into human embryonic kidney (HEK293) and Odora cells for promoter analysis studies. The Bright-Glo™ Luciferase Assay System was used to generate data from the study.  Results were presented as a comparison to pGL3-Control Vector transfectants. (2730)

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Proc. Natl. Acad. Sci. USA 100, 12153-12158. Genome-scale functional profiling of the mammalian AP-1 signaling pathway. 2003

Chanda, S.K., White, S., Orth, A.P., Reisdorph, R., Miraglia, L., Thomas, R.S., DeJesus, P., Mason, D.E., Huang, Q., Vega, R., Yu, D., Nelson, C.G., Smith, B.M., Terry, R., Linford, A.S., Yu, Y., Chirn, G., Song, C., Labow, M.A., Cohen, D., King, F.J., Peters, E.C., Schultz, P.G., Vogt, P.K., Hogenesch, J.B., and Caldwell, J.S.

Notes: A human cDNA library of ~20,000 sequences was tested for putative modulators of the activator protein-1 (Ap-1) signal transduction pathway. The plasmid library was co-transfected with luciferase reporter plasmids containing AP-1, p53 or Epo response elements. Transfections were performed in 384-well plates using HEK293, HCT116 or HepG2 cells. After 48 hours, the Bright-Glo™ Luciferase Assay was used to determine the level of luciferase activity in the transfections. Luminescence was read using an Acquest Plate Reader (LJL Biosystems) (3299)

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J. Virol. 77, 1992-2002. Molecular and functional analysis of an interferon gene from the zebrafish, Danio rerio. 2003

Altmann, S.M., Mellon, M.T., Distel, D.L. and Kim, C.H.

Notes: The pGEM®-T Easy Vector was used to subclone products of a 5´ RACE reaction. A promoter construct, assembled in the pGL3 Basic Vector, was co-transfected with a zebrafish interferon expression vector in the ZF4 zebrafish embryo fibroblast cell line using the TransFast™ Reagent (details provided). Luciferase levels were examined with the BrightGlo™ Luciferase Assay Reagent. Induction of zebrafish mRNA was also examined in zebrafish liver cells (ZFL) following treatment with the known interferon inducer, poly(I)-poly(C). RNA was extracted and reverse transcribed using ImProm-II™ Reverse Transcriptase. The resulting cDNA was used for quantitative, real-time RT-PCR with a SYBR green-based assay. (2627)

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Infect. Immun. 68 (11), 6402-6410. Identification of tgh-2, a filarial nematode homolog of Caenorhabditis elegans daf-7 and human transforming growth factor B, expressed in microfilarial and adult stages of Brugia malayi 2000

Gomez-Escobar, N., Gregory, W.F. and Maizels, R.M.

Notes: A neomycin resistance-expressing, luciferase reporter vector with an 800bp fragment of the 5´ end of the human plasminogen activator inhibitor-1 (PAI-1) gene was stably transfected into mink lung epithelial cells (MLECs).  The transfected MLECs were then infected with a TGH-2-expressing baculovirus and analyzed with the Bright-Glo™ Luciferase Assay System.  Relative Light Units from experimental samples were compared to an uninfected control. (2732)

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