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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. 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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Cell 121, 1097–108. Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development. 2005

Leaman, D., Chen, P.Y., Fak, J., Yalcin, A., Pearce, M., Unnerstall, U., Marks, D.S., Sander, C., Tuschl, T. and Gaul, U.

Notes: To monitor the effects of targeted degradation of micro RNA (miRNA) on Drosophila embryos, the authors cloned the full-length 3'UTRs of the proapoptotic factors hid and reaper into the psiCHECK™-2 Vector. The constructs were injected as plasmids (1µg/ml) mixed with 400µM sense and antisense miR-2 2'O-methyl oligoribonucleotides in early embryos. The total volume injected was equal to 5% of egg volume. After 10 hours development, the embryos were washed and lysed under agitation using 60µl lysis buffer and shaken at 750 rpm at room temperature for 30 minutes. The resulting lysate was cleared by centrifugation and three aliquots were tested using the Dual-Luciferase® Reporter Assay System. The Renilla-to-firefly luciferase ratios from three to five independent replicates were averaged and normalized to the value of the miR-6 sense control, the most severe apoptotic phenotype when targeted for depletion. Statistical significance was assessed using the t test. (3292)

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Proc. Natl. Acad. Sci. USA 102, 12759-12764. Biological function of the vaccinia virus Z-DNA-binding protein E3L: gene transactivation and antiapoptotic activity in HeLa cells. 2005

Kwon, J.A. and Rich, A.

Notes: The host gene E3L is required for vaccinia virus infection and has anti-apoptosis activity. The authors examine the ability of E3L to regulate transcription of several genes related to apoptosis, immune response and viral pathogenesis. IL-6, NF-AT, p53, NF-κB, Ap-1 and cAMP response elements were cloned upstream of a TATA box and the firefly luciferase reporter gene. Renilla luciferase (pRL-null Vector) was used to normalize for transfection efficiency. Luciferase activities were measured using the Dual Luciferase® Reporter Assay System. The authors also show that the Z-DNA binding region of E3L is important for transcriptional regulation. HeLa cells were transfected with an expression vectors expressing full-length E3L, E3L with a deletion of the Z-DNA binding domain or E3L with point mutations in residues important for Z-DNA binding. The AccessQuick™ RT-PCR System was used to quantitate IL-6, NF-AT and p53 mRNAs; β-actin was used as a control for RNA input. (3452)

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J. Biol. Chem. 280, 28215-28220. Determination of the functionality of common APOA5 polymorphisms. 2005

Talmud, P.J., Palmen, J., Putt, W., Lins, L., and Humphries, S.E.

Notes: The authors investigated common variants of the APOA5 gene that have been associated with differences in plasma triglyceride (TG) levels. PCR fragments containing either the –1131T --> C promoter variant or containing both the –1131T --> C and –3G --> A promoter variants were cloned into the pGEM®-T Vector System. The fragments were subsequently cloned into the pGL3 Basic Vector and transiently transfected into Huh7 and HepG2 cells along with the luciferase control vector, pRL-TK. The cells were lysed 48 hours after transfection and Luciferase activity was measured with the Dual-Luciferase® Reporter Assay System. The function of the 1891T --> C variant in the 3´ UTR was tested the same way; with the exception that site-directed mutagenesis was performed to introduce the T --> C at position 1891 before the fragment was cloned into the pGL3 Basic Vector. The functionality of the Kozak sequence –3A --> G variant was determined by cloning cDNAs into the pGEM®-7Zf Vector. Transcription/translation experiments were performed using the TNT® Quick Coupled Transcription/Translation System and the proteins were labeled using the FluorTect™ GreenLys System. In addition, a primer extension inhibition assay was performed using capped mRNAs generated with the Riboprobe® System –T7 and the Ribo m7G Cap Analog. Ribosome binding reactions were performed using the Rabbit Reticulocyte Lysate System, Nuclease Treated. (3460)

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Eukaryot. Cell 4, 1539-1549. Dual-luciferase assay system for rapid assessment of gene expression in Saccharomyces cerevisiae. 2005

McNabb, D.S., Reed, R., and Marciniak, R.A.

Notes: The firefly and Renilla luciferase coding regions were amplified from the pGL3 and pRL-CMV Vectors and cloned into various yeast expression vectors. Strains of Saccharomyces cerevisiae were transformed with these constructs and analyzed with the Dual-Luciferase® Reporter Assay System. The authors created yeast lysates for the Dual-Luciferase® Reporter Assay System using 1X Passive Lysis Buffer. Several factors important to assay performance as well as firefly and Renilla luciferase expression were explored, including the stability of both luciferases stored in lysates at room temperature for various periods of time, optimal culture density before lysis of transformants and the firefly luciferase half-life in S. cerevisiae. (3298)

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Plant Biotechnol. 22, 151–5. Multi-color luciferases as reporters for monitoring transient gene expression in higher plants 2005

Ogura, R., Matsuo, N., Wako, N., Tanaka, T., Ono, S. and Hiratsuka, K.

Notes: The authors evaluated use of a dual-color reporter assay for plant gene expression using the red and green light-emitting click beetle luciferase genes from the Chroma-Luc™ vectors (pCBR-Basic Vector, pCBG99-Basic Vector and pCBG68-Basic Vector). Plant expression vectors were constructed based on the reporter plasmid, pBI221, which contains the CaMV35S promoter, GUS reporter and nos-terminator cassette. The GUS cassette was replaced by the CBRluc, CBF99luc and CBR68luc genes. Twenty-five microliters of the gold microcarrier (1.6 mm) coated with 2µg plasmid DNA and were used to bombard plant specimens. Six hours after bombardment, an aqueous solution of 0.1mM D-luciferin potassium salt was sprayed on the transiently-transfected plants and luminescence detected using a CCD camera system. For the color-specific detection, the researchers used interference filters for wavelengths greater than 610nm (high-pass filter) and wavelengths between 510nm and 560nm (band-pass filter). To test luciferase activity in cell extracts, cultured tobacco (BY-2) and onion epidermal cells were cotransfected by bombardment using the click beetle constructs and a plasmid with the 35S promoter driving Renilla luciferase. The cells were homogenized in Passive Lysis Buffer and 8µl of the prepared lysate was assayed using the Dual-Luciferase® Reporter Assay. A second plasmid containing the inducible CAB1 promoter from Arabidopsis thaliana, was made by replacing the firefly luciferase gene with the CBG99luc-coding sequence from the pCBG99-Basic Vector. Spinach leaves were transfected by bombardment with the CAB1 construct, exposed to different light conditions for 21 hours and luminescence levels detected by CCD camera and interference filters. In all cases, the authors were able to detect click beetle luciferase expression in plants and cultured cells. (3293)

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Mol. Cell. Biol. 25, 6031–46. Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells. 2005

Chew, J.L., Loh, Y.H., Zhang, W., Chen, X., Tam, W.L., Yeap, L.S., Li, P., Ang, Y.S., Lim, B., Robson, P. and Ng, H.H.

Notes: The authors studied the effects of Embryonic Stem Cell (ESC)-specific regulation on the Pou5f1 promoter in human and mouse cells. To examine the effect of knockdown of Oct4 and Sox2 (two genes involved in ESC regulation) on the Pou5f1 promoter, a 3kb fragment of the human POU5F1 promoter was cloned into pGL3-Basic Vector and 100ng cotransfected with 100ng shRNA plasmids into mouse E14 ESCs. Five nanograms of pRL-SV40 Vector served as a transfection control. For the enhancer assay, a 461bp fragment of genomic DNA containing the SRR2 enhancer of Sox2 was amplified and cloned into the pGL3-Promoter Vector. The same amounts of plasmid, shRNA and transfection control were transfected into E14 ESCs as in the Pou5f1 promoter assay. To investigate gene knockdown in 293T cells, 5ng of the two open reading frame (ORF) constructs (the Luc-Sox2 and the Luc-Pou5f1 ORFs cloned into the psiCHECK™-2 Vector) were cotransfected with 100ng shRNA plasmid. The outcome was examined 48–60 hours post-transfection using the Dual-Luciferase® Reporter Assay System. (3291)

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J. Biol. Chem. 280, 38029–38034. Regulation of Oxidative Stress by the Anti-aging Hormone Klotho 2005

Yamamoto, M., Clark, J.D., Pastor, J.V., Gurnani, P., Nandi, A., Kurosu, H., Miyoshi, M., Ogawa, Y., Castrillon, D.H., Rosenblatt, K.P. and Kuro-o, M.

Notes: Mice that overexpress Klotho exhibit an extended lifespan and delayed aging. In this study, the authors show that Klotho protein protects against oxidative stress and activates the FoxO transcription factors, inducing expression of manganese superoxide dismutase. Two luciferase constructs were made, one with luciferase under the control of the Fas ligand promoter and one under the control of the human SOD2 promoter. HeLa cells were transfected with one of the two luciferase constructs and the pRL-CMV Renilla control vector. Transfected cells were treated with or without Klotho protein, and cell lysates were analyzed using the Dual-Luciferase® Assay. Klotho protein stimulated the activity of the Fas ligand gene promoter and the SOD2 promoter. (3667)

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Blood 105, 4685-4692. Src homology 2 domain-containing inositol-5-phosphatase 1 (SHIP1) negatively regulates TLR4-mediated LPS response primarily through a phosphatase activity- and PI-3K-independent mechanism. 2005

An, H., Xu, H., Zhang, M., Zhou, J., Feng, T., Qian, C., Qi, R. and Cao, X.

Notes: The authors of this study investigated the role of Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1) in Toll-like receptor 4 (TLR4)-mediated lipopolysaccharide (LPS) response. RAW264.7 macrophages were transfected with wildtype or mutant SHIP1 or control plasmid. Anti-ACTIVE® JNK and p38 antibodies were used in Western analyses to determine phosphorylation of these kinases in response to overexpression of SHIP1 or mutant SHIP1. To investigate any effects on IκB-alpha and NF-κB expression, RAW264.7 macrophages were cotransfected with pGL3-XκB-luciferase reporter plasmid and pRL-TK Renilla luciferase control plasmid. Transfected cells were treated with LPS for 6 hours or left untreated (control). The Dual-Luciferase® Reporter Assay System was used to monitor reporter gene expression. (3524)

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Nucl. Acids Res. 33, 4285-4310. Transcription factor binding sites in the pol gene intragenic regulatory region of HIV-1 are important for viral infectivity. 2005

Goffin, V., Demonté, D., Vanhulle, C., de Walque, S., de Launoit, Y., Burny, A., Collette, Y., Van Lint, C.

Notes: A fragment containing HIV-1 LAI 5' LTR was cloned into the unique EcoICRI-XhoI site of the pGL3-Basic Reporter Vector. The Luciferase Reporter Assay was used to analyze DNA transfected cells for luciferase activity. The pRL-TK Vector was used as a transfection efficiency internal control with a Renilla cDNA under control of HSV-TK. Firefly luciferase activity derived from the HIV-1 LTR was normalized to the Renilla luciferase activities using the Dual-Luciferase® Reporter Assay. (3703)

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J. Nutr. 135, 2987S-2992S. Zingiberaceous and citrus constituents, 1'-acetoxychavicol acetate, zerumbone, auraptene, and nobiletin, suppress lipopolysaccharide-induced cyclooxygenase-2 expression in RAW264.7 murine macrophages through different modes of action. 2005

Murakami, A., Shigemori, T. and Ohigashi, H.

Notes: These authors investigated the mechanisms by which anti-carcinogenic compounds derived from Japanese and subtropical vegetables and fruits attenuate LPS-induced COX-2 mRNA expression in RAW264.7 mouse macrophages. Using Western blot analysis, 10µg nuclear or 20µg cytosolic protein fractions isolated from LPS-treated macrophages in the absence or presence of various phytochemicals were stained with several antibodies for signal pathway proteins, including the Anti-ERK1/2 pAb. Further analysis of the MAPK and NF-κB systems was performed using firefly luciferase constructs co-transfected with the control pRL-TK Vector at a 1:1 ratio. Transfected cells were exposed to the plant compound for 12 hours and then to LPS for a further 12 hours. Reporter activity was measured using the Dual-Luciferase® Reporter Assay System. To determine the effect of the phytochemical zerumbone on the kinase reaction, a cell-free kinase assay was performed using Kinase-Glo® Assay System. In this assay, 1.5µl recombinant MAPKAPK-2, 0.2µl recombinant active p38α, 1µl zerumbone and 4µmol/l ATP were incubated for 3 hours at 28°C before addition of an equal volume of Kinase-Glo® Reagent. (3333)

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J. Biol. Chem. 279(10), 8787-8791. 14-3-3beta binds to big mitogen-activated protein kinase 1 (BMK1/ERK5) and regulates BMK1 function. 2004

Zheng Q., Yin G., Yan C., Cavet M. and Berk B.C.

Notes: The authors performed a yeast two-hybrid screen using big mitogen-activated kinase 1 (BMK1/ERK5) as the bait and identified the scaffolding protein 14-3-3beta. To confirm this interaction, the cloned mouse BMK1 gene was expressed in the TNT® T7 Quick Coupled Transcription/Translation System. The expressed protein was labeled with Transcend™ tRNA.  Using a GST-14-3-3beta fusion protein, a pull-down assay was performed and the direct binding confirmed after immunoblotting and staining with streptavidin-horseradish peroxidase (HRP).  The interaction of various BMK1 mutants were tested in a mammalian two-hybrid system and measured by the Dual-Luciferase® Reporter Assay System. (3078)

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