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J. Biol. Chem. 290, 30947-61. Chronic hyperinsulinemia causes selective insulin resistance and down-regulates uncoupling protein 3 (UCP3) through the activation of sterol regulatory element-binding protein (SREBP)-1 transcription factor in the mouse heart. 2015

Harmancey, R., Haight, D.L., Watts, K.A. and Taegtmeyer, H.

Notes: Differentiated L6 myocytes were seeded at 1 × 105 cells/well in a 24-well plate containing a 4:1 ViaFect™ Transfection Reagent: DNA ratio containing 1.025µg of DNA (reverse transfection). Cells were transfected with 500ng of a pGL4.12 [luc2CP]-based Ucp3 promoter construct, 25ng pGL4.74 [hRluc/TK] vector and 500ng of an SV-driven SREBP-1 expression vector. Luciferase activities were determined with the Dual-Luciferase® Reporter Assay System. The first intron was found to contain the cis-acting elements responsible for Ucp3 repression by SREBP-1. (4673)

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Cancer Res. 72, 810-820. SMYD3 Promotes Cancer Invasion by Epigenetic Upregulation of the Metalloproteinase MMP-9. 2012

Cock-Rada, A.M., Medjkane, S., Janski, N., Yousfi, N., Perichon, M., Chaussepied, M., Chluba, J., Langsley, G., and Weitzman, J.B.

Notes: These authors investigated the role of matrix metalloproteinase (MMP-9) in a reversible model of cancer that is initiated by infection with intracellular Theileria parasites. They found that gene induction by parasite infection was associated with trimethylation of histone H3K4 (H3K4me3) at the MMP-9 promoter. The H3K4 methyltransferase SMYD3 was the only histone methyltransferase upregulated upon infection. They therefore investigated the role of SMYD3 overexpression on MMP-9 expression and cell migration, identifying SMYD3 as an important new regulator of MMP-9 transcription. During the study they used the GloMax® Multi Luminometer to measure luminescence and absorbance in reporter and cell viability assays. They also used the Dual-Luciferase® Reporter Assay to measure SMYD3 activity in cells transfected with a SMYD3 reporter, and the pGL4-hRluc/TK plasmid for normalization of the experimental reporter activity. GoTaq® DNA polymerase was used in semi-quantitative RT-PCR. (4189)

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Nucl. Acids Res. 39(3), e16. Characterization of L1 retrotransposition with high-throughput dual-luciferase assays. 2011

Xie, Y., Rosser, J.M., Thompson, T.L., Boeke, J.D., and Wenfeng, A.

Notes: This paper describes a rapid dual-luciferase-based assay for L1 retrotransposition that is amenable to high-throughput screening. A firefly luciferase vector in which the luciferase gene was disrupted by an antisense intron was constructed by introducing a 900-bp fragment of the human γ-globin intron into pGL4.13. This Fluc gene, interrupted by an antisense intron, gives only minimal luciferase expression unless the luciferase gene is restored by a retrotransposition event. The authors also tested a similar retrotransposition reporter using the pGL4.73 Renilla luciferase vector, but found that the firefly construct gave much higher signals. They therefore used the firefly luciferase retrotransposition reporter, a Renilla luciferase normalization control and the Dual-Luciferase® Assay to characterize profiles of retrotransposition by various human and mouse L1 elements, and to measure the kinetics of L1 retrotransposition in cultured cells. The GloMax® Multi Luminometer was used to quantify luciferase activity. (4205)

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J. Virol. 81, 558–567. Hijacking components of the cellular secretory pathway for replication of poliovirus RNA. 2007

Belov, G.A., Altan-Bonnet, N., Kovtunovych, G., Jackson, C.L., Lippincott-Schwartz, J. and Ehrenfeld, E.

Notes: In this study, the Renilla luciferase gene from the phRL-CMV Vector was incorporated into polioviral RNA in place of a structural protein-coding region. Renilla luciferase activity was then monitored as a convenient indicator of viral replication. Specifically, these authors tested whether the guanine nucleotide exchange factors GBF1 and BIG2 could rescue brefeldin A (BFA)-induced inhibition of polioviral replication in HeLa cells. Cells were transfected with plasmids encoding either GBF1 or BIG2, together with the firefly-luciferase-encoding pGL4.13 Vector, which was used as a control to monitor transfection efficiency. Eighteen hours post-transfection, the cells were transfected with polioviral replicon RNA containing the Renilla luciferase gene. One hour later, normal growth medium containing EnduRen® Live Cell Substrate and either BFA or solvent alone, was added and Renilla luciferase activity was monitored at hourly intervals for 7 hours. Cells were then lysed and firefly luciferase activity was measured to assess transfection efficiency using the Dual-Glo® Luciferase Assay System System. In the presence of BFA, GBF1 was able to partially rescue viral replication. In the presence of BIG2 and BFA, no viral replication occurred. (3562)

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Nucl. Acids Res. 34, 6640–6652. Brn-3b enhances the pro-apoptotic effects of p53 but not its induction of cell cycle arrest by cooperating in trans-activation of bax expression. 2006

Budhram-Mahadeo, V.S., Bowen, S., Lee, S., Perez-Sanchez, C., Ensor, E., Morris, P.J. and Latchman, D.S.

Notes: Previously, the POU domain of Brn-3a was shown to interact with p53 and increase cell survival. In this article, the authors explored the possibility that Brn-3b, which shares a POU domain 95% identical to Brn-3a, may interact with p53 and affect its role in apoptosis. To test the protein:protein interaction, GST-Brn-3b fusion protein was bound to glutathione Sepharose beads and incubated with 35S-methionine labeled full-length or truncated p53, prepared using the TNT® T7 rabbit reticulocyte lysate. The luciferase control included in the kit was used as the noninteracting protein control. After washing, the bound proteins were resolved by 12% SDS-PAGE, and the bands examined by radiography. To examine the effect of Brn-3b on two p53-regulated genes, Bax and p21cip1/waf1, ND7 cells were transiently transfected with empty vector, Brn-3b, Brn-3a or p53, or Brn-3 with p53. The reporter gene cotransfected was under the control of wildtype Bax, wildtype p21cip1/waf1, or mutant Bax. A control vector (Renilla luciferase driven by the thymidine kinase promoter) was used for normalization. Forty-eight hours posttransfection, the cells were harvested and reporter levels assessed using the Dual-Luciferase® Reporter Assay System. (3597)

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J. Clin. Oncol. 24, 983-7. Elevated serum B-lymphocyte stimulator levels in patients with familial lymphoproliferative disorders. 2006

Novak, A.J., Grote, D.M., Ziesmer, S.C., Kline, M.P., Manske, M.K., Slager, S., Witzig, T.E., Shanafelt, T., Call, T.G., Kay, N.E., Jelinek, D.F., Cerhan, J.R., Gross, J.A., Harder, B., Dillon, S.R. and Ansell, S.M.

Notes: To test the significance of the C or T polymorphism at position -871 of the serum B-lymphocyte stimulator (BLyS) promoter region, the BLyS promoter was amplified, digested with Kpn I and cloned into the pGL3-Enhancer Vector. HL60 cells were then transiently transfected by electroporation using 10µg of the pGL3-BLyS-promoter constructs and 40ng of the pGL4.75[hRluc/CMV] Vector, which expresses Renilla luciferase. After 48 hours, expression of the reporter genes was assessed using the Dual-Luciferase® Reporter Assay System. (3343)

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