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Cancer Res. 67, 1979-1987. Twist transcriptionally up-regulates AKT2 in breast cancer cells leading to increased migration, invasion and resistance to paclitaxel. 2007

Cheng, G.Z., Chan, J., Wang, Q., Zhang, W. and Wang, L-H.

Notes: To study the molecular mechanism underlying metatasis, the authors established a model system to select highly invasive cells. Increased Twist and AKT2 expression was noted in the highly invasive cells and the authors sought a functional connection between these two proteins. HEK293T cells were transfected with AKT2-Luc, a control vector encoding Renilla luciferase, and increasing amounts of Myc-Twist. The Dual-Luciferase® Reporter Assay System was used to measure firefly luciferase activity, normalized to Renilla activity to determine whether Twist was able to transactivate full-length AKT2 promoter. The results, as measured by luciferase activity, showed that Twist led to a dosage-dependent increase in AKT2 promoter transactivation. (3605)

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J. Leukoc. Biol. 79, 628-638. Mycobacterium paratuberculosis, Mycobacterium smegmatis, and lipopolysaccharide induce different transcriptional and post-transcriptional regulation of the IRG1 gene in murine macrophages. 2006

Basler, T., Jeckstadt, S., Valentin-Weigand, P., and Goethe, R.

Notes: In this study a 984bp fragment of the IRG1 5´ promoter region was cloned into the pGL3 Basic Vector. Transfection-quality plasmid DNA was purified using the PureYield™ Plasmid Midiprep System and used to transfect RAW264.7 cells. Twenty-four hours post-transfection, cells were stimulated with LPS or infected with Mycobacterium paratuberculosis or Mycobacterium smegmatis for an additional 24 hours. Relative luciferase activities in LPS-stimulated and infected macrophages were then assayed using the Dual-Luciferase® Reporter Assay System. (3365)

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Cancer Res. 66, 7151-7157. Absence of the full-length breast cancer-associated gene-1 leads to increased expression of insulin-like growth factor signaling axis members. 2006

Shukla, V., Coumoul, X., Cao, L., Wang, R.-H., Xiao, C., Xu, X., Andò, S., Yakar, S., LeRoith, D. and Deng, C.

Notes: The authors of this study investigated the influence of BRCA1 on insulin-like growth factor 1 (IGF-1) signaling. In mice lacking full-length BRCA, they observed increased IGF-1 expression as well as changes in expression of other proteins within the IGF-1 signaling pathway including Irs-I. They observed increases in IGF-I and Irs-I expression in mammary tumors from these same mice. To understand better the relationship between BRCA1 and IGF, they transfected a mouse mammary tumor cell line with a small hairpin RNA directed against BRCA1 and showed that Irs-1 mRNA and promoter activity increases. Similar results were observed in human UBR60 cells. Irs-1 promoter activity was assessed using the Dual-Luciferase® Reporter Assay System. (3604)

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J. Biol. Chem. 281, 14700–14710. Attenuation of peroxisome proliferator-activated receptor gamma (PPARgamma) mediates gastrin-stimulated colorectal cancer cell proliferation. 2006

Chang, A.J., Song, D.H. and Wolfe, M.M.

Notes: The researchers sought to determine whether PPARγ expression might direct the effects of gastrin in proliferation of colorectal cancer cells (CRC). They determined that cell line DLD-1 cells had both PPARγ and gastrin receptors. They demonstrated that gastrin stimulated CRC cell proliferation with a coincident decrease in PPARγ levels. These studies show that gastrins trophic properties could be due in part to transactivation of EGFR and phosphorylation of ERK1/2, leading to a decrease in PPARγ activation.

The authors used CellTiter 96® Aqueous One Solution Cell Proliferation Assay to measure cell growth of the CRC cell line DLD-1.

The DLD-1 cells were transiently transfected with a luciferase vector, and FuGENE® 6 Transfection Reagent was used at a DNA ratio of 3:1 in 24-well plates. To normalize for transfection efficiency, the cells were co-transfected with a β-Gal reporter construct. The Dual-Luciferase® Assay System was used to measure PPARgamma activity with values then normalized to Beta-Gal, measured with the β-Galactosidase Enzyme Assay System.  (4280)

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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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Circ. Res. 98, 532–539. Cellular senescence impairs circadian expressionof clock genes in vitro and in vivo 2006

Kunieda, T., Minamino,T., Katsuno, T., Tateno,K., Nishi, J-i., Miyauchi, H., Orimo, M., Okada, S. and Komuro, I.

Notes: The authors of this study investigated the effect of cellular senescence on expression of the genes that regulate circadian rhythms, specifically Per2 and BmalI. The Dual-Luciferase® Assay was used to determine activity of a PER gene reporter in senescent and young primary cultured human aortic vascular smooth muscle cells. Senescent cells activated CREB, but much more weakly than dividing cells did. (3672)

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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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J. Immunol. 177, 147–154. Flotillin-1 regulates IgE receptor-mediated signaling in rat basophilic leukemia (RBL-2H3) cells. 2006

Kato, N., Nakanishi, M. and Hirashima, N.

Notes: The CheckMate™ Mammalian Two-Hybrid System was used to study the association of Lyn, a tyrosine kinase, and flotillin-1, a novel constituent of lipid rafts. NIH/3T3 cells were seeded in 60mm dishes at a density of ~1 × 105 cells. Each plate of cells was transfected with a total of 5.4µg of plasmid DNA (a 1:1:1 mixture of pBIND-flotillin-1 vector, pACT-Lyn vector, and pG5luc vector) by lipofection. Both mutant and wildtype proteins were used to assess the interaction. The cells were harvested, lyses and assessed for firefly and Renilla luciferase activities using the Dual-Luciferase® Reporter Assay System. The results were expressed as a ratio of firefly to Renilla luciferase activity. (3491)

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J. Virol. 80, 6669-6677. Human papillomavirus e7 oncoprotein dysregulates steroid receptor coactivator 1 localization and function.
2006

Baldwin, A., Huh, K-W. and Mϋnger, K.

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. (3459)

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Nucl. Acids Res. 34, e107. Identification of miRNA targets with stable isotope labeling by amino acids in cell culture. 2006

Vinther, J., Hedegaard, M.M., Gardner, P.P., Andersen, J.S. and Arctander, P.

Notes: These authors used stable-isotope labeling of amino acids in culture (SILAC), a method that allows quantitation of relative protein abundance between populations, to investigate the effect of the microRNA miRNA-1 on the HeLa cell proteome. HeLa cells grown in the presence of labeled arginine and lysine were transfected with miRNA-1, and the labeled proteins compared to those from control cells treated with the transfection reagent alone. A set of 16 proteins repressed by miRNA-1 was identified. The 3´ UTR's from 11 of the miRNA-1-regulated genes were tested in a reporter assay, and 6 were shown to repress expression in an miRNA-1-dependent fashion. For the reporter assays, the HSV TK promoter was amplified from the pRL-TK Vector and subcloned into the pGL4.12(luc2CP) Vector, creating the pGL4.12-TK vector. The 3´ UTR regions from suspected target genes were then amplified and subcloned into the pGL4.12-TK construct. The various pGL4.12-TK constructs (0.9µg) were then co-transfected along with pRL-TK (0.1µg) and miRNA-1 (50pmol) into HeLa cells (80,000 cells/well in 12-well plates). Twenty-two hours post-transfection, firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. (3561)

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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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Mol. Cell. Biol. 26, 4652–63. MafG sumoylation is required for active transcriptional repression. 2006

Motohashi, H., Katsuoka, F., Miyoshi, C., Uchimura, Y., Saitoh, H., Francastel, C., Engel, J.D., and Yamamoto, M.

Notes: The authors examined the role of sumoylation in transcriptional repression by the homodimeric MafG protein and in transcriptional activation by a MafG/p45 heterodimer. To monitor transcription levels in the presence of MafG and mutated forms of MafG, the authors cotransfected 293T cells with a plasmid containing three copies of a Maf recognition site (MARE) driving expression of the firefly luciferase reporter gene, and an unspecified plasmid with the Renilla luciferase reporter gene for normalization. Reporter gene activities were measured using the Dual-Luciferase® Reporter Assay System. To examine DNA binding ability, polyhistidine-tagged MafG and MafG mutants were expressed in the TNT® Coupled Wheat Germ Extract System and used in electrophoretic mobility shift assays. The DNA-binding ability was also examined using biotinylated double-stranded DNA probes with a MARE sequence. The MafG proteins were allowed to bind to this probe, and the protein-DNA complexes were captured using the TetraLink™ Tetrameric Avidin Resin then separated by SDS-PAGE. (3660)

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Cancer Res. 66, 9090-9098. MicroRNA regulates the expression of human cytochrome P450 1B1. 2006

Tsuchiya, Y., Nakajima, M., Takagi, S., Taniya, T., and Yokoi, T.

Notes: These authors identified a region complementary to the microRNA miR-27b in the 3´ UTR of the cytochrome p450 CYP1B1 mRNA, and showed that miR-27b was involved in regulation of CYP1B1 expression. The 3´ UTR containing the miRNA target site was cloned downstream of the luciferase gene in the pGL3 Promoter Vector and cotransfected into the miR-27b-positive breast cancer cell line MCF-7 and into miR-27b-negative Jurkat cells. Luciferase expression levels from the reporter vector containing the CYP1B1 3´ UTR sequence were reduced in miR-27b-positive cells, but not in the Jurkat cell controls. Delivery of an antisense oligoribonucleotide directed against miR-27b to MCF-7 cells containing the reporter construct resulted in restoration of luciferase activity. The effects of inhibition of miR-27b on protein levels and enzymatic activity of CYP1B1 were then investigated in MCF-7 cells. CYP1B1 protein levels and enzymatic activity increased significantly in cells transfected with the antisense oligo; the enzymatic activity was measured using a p450-Glo™ Assay. The coding region and 3´ UTR of the CYP1B1 gene were also PCR-amplified, subcloned the into the pTargeT™ Mammalian Expression Vector, and transfected into HEK293 cells. The effect of overexpression of miR-27b on protein levels and enzymatic activity of CYP1B1 was then evaluated in these cells. (3622)

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Proc. Natl. Acad. Sci. USA 103, 15669–15674. miR-7b, a microRNA up-regulated in the hypothalamus after chronic hyperosmolar stimulation, inhibits Fos translation. 2006

Lee, H-J, Palkovits, M. and Young, W.S.

Notes: In this study, the psiCHECK™ Vector was used in an investigation of the interaction between a microRNA and its potential target mRNA. Increased Fos expression in the paraventricular (PVN) and supraoptic (SON) nuclei is associated with hyperosmolality. In an effort to identify microRNAs that may regulate Fos expression, miRNA was isolated from the PVN and SON of mice after 10 days of 2% saline ingestion, and differentially expressed miRNAs were identified by microarray analysis. The miR-7b miRNA, which was overexpressed after saline treatment, was selected for further analysis as the fos gene 3´ UTR contains putative miR-7b binding sites. To investigate the potential interaction between miR-7b and Fos, the 3´ UTR of fos was subcloned downstream of the Renilla luciferase gene in the psiCHECK™ Vector. 293T cells were then co-transfected with the psiCHECK-Fos vector construct (0.8µg) and a vector expressing miR-7b and GFP (2.4µg). Luciferase assays were performed 42 hours post-transfection using the Dual-Luciferase® Reporter Assay System. A 40% reduction in Renilla expression was observed in cells co-transfected with the miR-7b vector compared with cells transfected with psiCHECK-Fos and a control miRNA. (3560)

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Nucl. Acids Res. 34, 485-495. Nucleolin links to arsenic-induced stabilization of GADD45alpha mRNA. 2006

Zhang, Y., Bhatia, D., Xia, H., Castranova, V., Shi, X. and Chen, F.

Notes: The induction of GADD45α (growth arrest and DNA damage inducible gene 45α) in response to arsenic was examined on the protein and mRNA levels. Protein levels were determined by Western blotting; mRNA levels were determined using the AccessQuick™ RT-PCR System. Changes in GADD45α promoter activity in response to arsenic treatment were monitored in cells transiently transfected with constructs containing GADD45α promoter elements upstream of the firefly luciferase reporter gene. The Dual-Luciferase® Assay System was used to quantitate luciferase expression, and thus, promoter activity. (3440)

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Mol. Cell. Biol. 26, 4934-4348. Platelet-derived growth factor BB induces nuclear export and proteasomal degradation of CREB via phosphatidylinositol 3-kinase/Akt signaling in pulmonary artery smooth muscle cells. 2006

Garat, C.V., Fankell, D., Erickson, P.F., Reusch, J.E.-B., Bauer, N.N., McMurty, I.F., and Klemm, D.J.

Notes: cAMP/PKA signaling appears to be involved in restraining cell proliferation in healthy pulmonary arteries. Expression of CREB, a transcription factor that is a primary target of PKA activity, is decreased in proliferating smooth muscle cells (SMCs) of pulmonary arteries. This study investigated the regulatory mechanisms and signaling pathways that determine the levels of CREB in SMCs. Pulmonary arteries were harvested from adult rat lungs, and SMCs were obtained and cultured. SMCs at passages 1-8 were used for studies. Proliferation of the SMCs was measured using the CellTiter® 96 AQueous Non-Radioactive Cell Proliferation Assay. Total and PDGF-stimulated PKA and PKC activities were measured using in the PepTag® Non-Radioactive cAMP-Dependent and PKC Protein Kinase Assays. SMCs transfected with a plasmid containing a CREB-responsive promoter linked to the firefly luciferase gene were treated with PDGF or inhibitors. As a control, cells were cotransfected with a Renilla luciferase reporter plasmid. A Dual-Luciferase® Assay was performed to determine CREB transcriptional activity. (3485)

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J. Biol. Chem. 281, 2044–2052. Proline oxidase, a proapoptotic gene, is induced by troglitazone: evidence for both peroxisome proliferator-activated receptor gamma-dependent and -independent mechanisms. 2006

Pandhare, J., Cooper, S.K. and Phang, J.M.

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. (3514)

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J. Biol. Chem. 280, 28412-28423. Protein Kinase C βII plays an essential role in dendritic cell differentiation and autoregulates its own expression. 2006

Cejas, P.J., Carlson, L.M., Zhang, J., Padmanabhan, S., Kolonias, D., Lindner, I., Haley, S., Boise, L.H. and Lee, K.P.

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. (3407)

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J. Immunol. 176, 5519-5528. 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. 2006

Perez, L.E., Chandrasekar, B., Saldarriaga, O.A., Zhao, W., Arteaga, L.T., Travi, B.L. and Melby, P.C.

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 (3470)

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J. Biol. Chem. 281, 14691–14699. Regulation of Aurora-A kinase gene expression via GABP recruitment of TRAP220/MED1. 2006

Udayakumar, T.S., Belakavadi, M., Choi, K.H., Pandey, P.K. and Fondell, J.D.

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. (3607)

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J. Immunol. 176, 5050–5059. Substance P stimulates cyclooxygenase-2 and prostaglandin E2 expression through JAK-STAT activation in human colonic epithelial cells. 2006

Koon, H.W., Zhao, D., Zhan, Y., Rhee, S.H., Moyer, M.P. and Pothoulakis, C.

Notes: To examine the effect of Substance P (SP) on COX-2 expression, the COX-2 promoter region spanning –2069 to –66 bp was cloned by PCR and subcloned into the pGL3-Basic Vector (pGL3-Cox-2). Nontransformed human colonic epithelial NCM460 cells overexpressing neurokinin-1 receptor (NK-1R; NCM460-NK-1R) were seeded in 12-well plates and transiently transfected with pGL3-Cox-2 with either a transfection control pRL-TK Vector or siRNA or both. The siRNA molecules used were for AK1, JAK2 (Upstate Biotechnology), STAT3, STAT5, STAT6 or a control siRNA. The transfected cells were serum starved for 24 hours, treated with SP for 4 hours and then lysed. The cell extracts were measured for firefly and Renilla luciferase activities using the Dual-Luciferase® Reporter Assay System. Relative luciferase activity was a ratio of COX-2 promoter firefly activity to Renilla activity; results were expressed as percentage of control group without SP stimulation. To mutate the STAT binding elements, the pGL3-Cox-2 construct was modified using the GeneEditor™ in vitro Site-Directed Mutagenesis System. The resulting mutant constructs were tested in the same system as the wildtype COX-2 promoter. (3520)

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J. Immunol. 176, 5050-9. Substance P stimulates cyclooxygenase-2 and prostaglandin E2 expression through JAK-STAT activation in human colonic epithelial cells. 2006

Koon, H-W., Zhao, D., Zhan, Y., Rhee, S.H., Moyer, M.P. and Pothoulakis, C.

Notes: The COX-2 promoter was cloned by PCR into the pGL3 Vector. NCM460-NK-1R cells were transiently transfected with the cloned promoter and the pRL-TK vector as an internal control or siRNA targeted against various JAK/STAT genes. The Dual-Luciferase® Assay was used to measure promoter activity. The wildtype COX-2 promoter was mutagenized using the GeneEditor™ in vitro Site-Directed Mutagenesis System. (3384)

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J. Biol. Chem. 281, 17635–17643. 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. 2006

de Wolf, C.J., Cupers, R.M., Bertina, R.M. and Vos, H.L.

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. (3510)

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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. 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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