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J. Cell Sci. 121, 3207–3217. All EGF(ErbB) receptors have preformed homo- and heterodimeric structures in living cells. 2008

Tao, R.H. and Maruyama, I.N.

Notes: The CheckMate™ Mammalian Two-Hybrid System was used to explore the dimerization of epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases without ligand. The intracellular domains of EGFR, ErbB2, ErbB3 and ErbB4 were amplified and cloned into both the pACT and pBIND Vectors. Transfection into NIH3T3 cells in 12-well plates occurred with 0.3 μg of pG5luc Vector (the reporter vector), 0.2 μg of pACT Vector or an equimolar amount of pACT construct, and 0.1 μg of pBIND Vector or an equimolar amount of pBIND construct. After 24 hours, the cells were lysed and luciferase activity assessed using the Dual-Luciferase® Reporter Assay System. (3993)

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J. Biol. Chem. 283, 5719–27. Avian and 1918 Spanish influenza a virus NS1 proteins bind to Crk/CrkL Src homology 3 domains to activate host cell signaling. 2008

Heikkinen, L.S., Kazlauskas, A., Melén, K., Wagner, R., Ziegler, T., Julkunen, I. and Saksela, K.

Notes: The authors identified the N-terminal Src homology 3 (SH3) domain-binding motif of Crk and CrkL as the preferred binding partner of nonstructural protein 1 (NS1), an important virulence factor of the influenza A virus. Interaction of NS1 with Crk, CrkL and other SH3 domain-containing proteins p85, p85β and Eps8L1 was investigated by protein pull-down assays. Expression constructs for biotinylated Crk, CrkL, p85, p85β and Eps8L1 were created by amplifying the 123 amino acid biotin acceptor domain from Propionibacterium shermanii from the PinPoint Xa-T Vector and inserting it upstream of the protein-coding sequences in a pGEX vector derivative. These constructs and a construct encoding Myc-tagged NS1 were transfecting into 293FT cells, and the biotinylated proteins were immobilized from the cell lysate using TetraLink™ Tetrameric Avidin Resin. Any Myc-NS1 that bound to the immobilized protein was detected using Western Blot analysis and an anti-Myc antibody. The authors also investigated the ability of wildtype NS1 or NS1 mutants to inhibit interferon-induced gene expression. A reporter plasmid was created by cloning an interferon-stimulated response element upstream of a minimal thymidine kinase promoter driving firefly luciferase expression. A vector containing Renilla luciferase was used as a control to normalize transfection efficiency. Huh-7 cells were cotransfected with the firefly and Renilla luciferase reporter constructs (0.2µg and 5ng, respectively), treated with interferon-β and lysed using the Passive Lysis Buffer. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay System. (3803)

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Development 135, 541-557. Impaired generation of mature neurons by neural stem cells from hypomorphic Sox2 mutants 2008

Cavallaro, M., Mariani, J., Lancini, C., Latorre, E., Caccia, R., Gullo, F., Valotta, M., DeBiasi, S., Spinardi, L., Ronchi, A., Wanke, E., Brunelli, S., Favaro, r., Ottolenghi, S. and Nicolis, S.K.

Notes: The authors of this paper investigated the role of Sox2 in neuronal differentiation. Neurospheres were derived from the brains of normal and Sox2 hyphomorphic mice and used to generate differentiated neural cells. In astroglia from cultures containing a Sox2-GFP-expressing lentivirus, ectopic expression of Sox2 correlated with reduced GFAP expression. The authors investigated the role of Sox2 by amplifying binding sites upstream of the GFAP promoter and cloning them upstream of the thymidine kinase promoter in the pRL-TK vector. The Dual-Luciferase Assay System was used to analyze the effect of Sox2 expression on the luciferase reporter gene. The DeadEnd™ Fluorometric TUNEL System was also used to assess apoptosis in some of the neurosphere-derived cultures. (3953)

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Biol. Reprod. 79, 938–946. Japanese eel follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh): production of biologically active recombinant Fsh and Lh by Drosophila S2 cells and their differential actions on the reproductive biology. 2008

Kazeto, Y., Kohara, M., Miura, T., Miura, C., Yamaguchi, S., Trant, J.M., Adachi, S. and Yamauchi, K.

Notes: The ORFs of Japanese eel follicle-stimulating hormone receptor (Fshr) and luteinizing hormone receptor (Lhr) were ligated into the pSI Mammalian Expression Vector. Fifty nanograms of this construct or the pSI Vector was cotransfected with 0.5ng of pRL-null Vector and 1µg or a cAMP-responsive firefly luciferase reporter vector into COS cells seeded in 60mm dishes. The next day, the cells were trypsinized, replated into 96-well plates and allowed to recover for 1 day. The transfected cells were serum-starved then treated with hormones six hours before being lysed and enzymatic activity measured with the Dual-Luciferase® Reporter Assay System. (3988)

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Molecular Pharmacology Fast Forward March 11, 2008, epub ahead of print. Kavalactones protect neural cells against amyloid β peptide-induced neurotoxicity via ERK1/2-dependent Nrf2-activation 2008

Wruck, C.J., Götz, M.E., Herdegen, T., Varoga, D., Brandenburg, L-O. and Pufe, T.

Notes: The accumulation of the toxic form of the Amyloid-β peptide is known to induce oxidative damage in the brain. Although treatment with antioxidants has not proven effective at controlling AD symptoms, inducing the natural systems in the brain that protect from oxidative damage may provide a possible therapeutic approach. A host of antioxidant and detoxifying enzymes are upregulated by binding of the Nrf2 transcription factor to the ARE (antioxidant response element) regulatory sequence. The authors used a Dual Luciferase® Reporter Assay to assess modulation of gene activity through ARE by kavalactones. Kavalactones are compounds found in the roots and rhizomes of Kava (Piper methysticum), a plant cultivated an used in some Pacific societies for medicinal and social uses. The ARE1 region from the rat NAD(P)H:quinone oxidoreductase-1 gene was placed upstream of a pGL3 firefly luciferase reporter construct and cotransfected along with a pRL-TK Renilla control construct into PC12 or C6 cells. The data show induction of luciferase activity by kavalactones. Further investigation shows that the kavalactones promote Nrf2 stabilization possibly through the ERK1/2 pathway. (3859)

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Nucl. Acids Res. 36, 5391–404. miR-16 family induces cell cycle arrest by regulating multiple cell cycle genes. 2008

Liu, Q., Fu, H., Sun, F., Zhang, H., Tie, Y., Zhu, J., Xing, R., Sun, Z. and Zheng, X.

Notes: To identify microRNA targets, the authors created a Drosha-knockdown cell line and confirmed depletion of Drosha and three randomly selected miRNAs in these cells by quantitative RT-PCR, using β-actin as a control. The reverse transcription step was performed using the ImProm-II™ Reverse Transcription System. The authors then performed microarray analysis to monitor expression of transcripts to determine which were upregulated as a result of Drosha depletion; cRNA used in these microarray experiments was synthesized using the T7 RiboMAX™ Express Large Scale RNA Production System. Cyclin D1 was identified as a potential miRNA target. To screen miRNAs that regulate cyclin D1, the authors cloned the cyclin D1 3´ untranslated region downstream of the firefly luciferase gene of the pGL3-Control Vector and measured luciferase levels in transfected cells using the Dual Luciferase Reporter Assay System. Renilla luciferase in the pRL-TK Vector was used as a normalization control. (3894)

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Mol. Pharmacol. 73, 1113-1121. PPP1R16A, the membrane subunit of protein phosphatase 1{beta}, signals nuclear translocation of the nuclear receptor CAR. 2008

Sueyoshi, T., Moore, R., Sugatani, J., Matsumura, Y. and Negishi, M.

Notes: This article examines the interaction between CAR, a member of the nuclear steroid/thyroid hormone receptor family, which translocates from the cytoplasm to the nucleus when cells are exposed to phenobaritol, and the membrane-associated subunit of protein phosphatase 1 (PPP1R16A, or abbreviated as R16A), which is a novel CAR-binding protein. The R16A protein, it was expressed using the TnT® Coupled Reticulocyte Lysate System and labeled with 35S. This protein was incubated with GST-hCAR-fusion protein attached to a glutathione resin; the resin was washed, and the bound protein was separated by PAGE and detected by autoradiography. Affinity-tagged R16A was expressed in HepG2 cells, purified and separated by SDS-PAGE. The two major bands were excised, digested with Sequencing Grade Modified Trypsin, lyophilized, resuspended in acetonitrile and subjected to mass spectrometric analyses. The CheckMate™ Mammalian Two-Hybrid System was used to examine the interaction between wildtype and mutated R16A. Forty-eight hours posttransfection into HepG2 cells or 16 hours after DNA injection into mice and liver homogenization, the luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. (3752)

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J. Biol. Chem. 283, 23514–23. Snail regulates cell-matrix adhesion by regulation of the expression of integrins and basement membrane proteins. 2008

Haraguchi, M., Okubo, T., Miyashita, Y., Miyamoto, Y., Hayashi, M., Crotti, T.N., McHugh, K.P. and Ozawa, M.

Notes: Snail is a transcriptional repressor of E-cadherin that enhances both cell attachment and cell detachment in Madin Darby canine kidney (MDCK) and A4231 cells. To investigate this effect, the authors used Western blot analysis and RT-PCR to monitor protein and mRNA levels of the major adhesive proteins expressed in epithelial cells: laminin, heparin sulfate proteoglycan and collagens. For RT-PCR, total RNA was isolated from transiently transfected snail-expressing MDCK and A431 cells and untransfected cells, then reverse transcribed. The resulting cDNA was amplified by PCR using GoTaq® DNA Polymerase; glyceraldehyde-3-phosphate dehydrogenase was amplified as an internal control. The ability of Snail to regulate the integrin αV promoter was also examined by cloning the promoter and several promoter deletions upstream of a firefly luciferase reporter gene in the pGL3-Basic Vector. Each of these constructs (1µg) and 20ng of pRL-CMV Vector were transfected into MDCK and MDCK/snail cells, and luminescence was measured using the Dual Luciferase Assay System. (3882)

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Blood 111, 33498-33506. Transcription factor Erg regulates angiogenesis and endothelial apoptosis through VE-cadherin. 2008

Birdsey GM, Dryden NH, Amsellem V, Gebhardt F, Sahnan K, Haskard DO, Dejana E, Mason JC, Randi AM.

Notes: These authors showed that the ETS transcription factor Erg interacts with the VE-cadherin promoter region and regulates endothelial apoptosis through this interaction. They demonstrated that inhibition of Erg by siRNA resulted in decreased VE-cadherin mRNA and protein levels, and showed that Erg interacts with the VE-cadherin promoter using a CHIP assay. To show the functional relevance of this interaction, HeLa cells were transfected with a pGL4 Vector containing the VE-cadherin promoter region and an expression vector containing Erg2 cDNA. In this reporter assay, Erg overexpression resulted in ~1.8 fold transactivation of VE-cadherin promoter activity, as measured using the Dual-Luciferase® Reporter Assay System. Inhibition of Erg in human umbilical vein endothelial cells also resulted in a loss of viability and an increase in activation of caspase 3 and caspase 7. The authors showed that apoptosis could be significantly decreased by overexpression of VE-cadherin, indicating that Erg regulates survival partially via its interaction with VE-cadherin. The Caspase-Glo® 3/7 Assay was used to measure caspase activity in these experiments. (3872)

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Anticancer Res. 27, 3843–8. Pamidronate down-regulates urokinase-type plasminogen activator expression in PC-3 prostate cancer cells. 2007

Iguchi, K. et al.

Notes: In this paper, the authors hypothesized that bisphosphonates, which are used to prevent tumor metastasis, affect expression of urokinasetype plasminogen activator (uPA), which seems to be critical for prostate cancer metastasis. The authors examined the effect of several bisphosphonates on uPA expression in PC-3 cells. Pamidronate treatment resulted in lower uPA mRNA levels. To investigate the cause, the authors created a uPA reporter construct (pGL3-uPA) by cloning the 5′-flanking region of the human uPA gene upstream of a firefly luciferase reporter gene in the pGL3-Basic Vector. PC-3 cells were seeded at a density of 3 × 104 cells/well in 24-well culture plates and transfected with 0.5µg of pGL3-uPA and 1ng of the Renilla luciferase phRL-TK Vector using FuGENE® 6 Transfection Reagent. At 48 hours post-transfection, the authors measured reporter activity using the Dual-Luciferase® Reporter Assay System to learn that treatment with 100µM pamidronate inhibited transcription of the uPA gene. (4384)

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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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Mol. Pharmacol. 72, 1380–1390. Activation of Nrf2 by toxic bile acids provokes adaptive defense responses to enhance cell survival at the emergence of oxidative stress. 2007

Tan, K.P., Yang, M. and Ito, S.

Notes: The authors explored the role that nuclear factor (erythroid 2-like) factor 2 (Nrf2) may have in mitigating the cytotoxic effects of bile acids on cells. A reporter vector was constructed using the core sequence of antioxidant reporter element (ARE) sythnesized by annealing two complementary oligonucleotides with Kpn1 and BglII at the 5’ and 3’ ends, respectively, and ligated into the same restriction sites of the pGL3-Promoter Vector (designated pGL3_ARE). To ensure specificity for the experiments, three point mutations were introduced into the ARE sequence (designated pGL3_mARE). To create HepG2 cells that stably expressed human Na(+)-taurocholate co-transporting polypeptide (NTCP), the cDNA clone of NTCP was subcloned into pTargeT™ Mammalian Expression Vector via the NotI site and selected using 500μg/ml G-418. The stable clones or standard HepG2 cells were transiently transfected with 0.1µg of pGL3_ARE or pGL3_mARE, 0.02µg of the control reporter pRL-TK Vector, with or without Nrf2 or dominant negative Nrf2 expression constructs. After overnight transfection, the cells were treated with bile acids for 16–18 hours and luciferase activities determined using the Dual-Luciferase® Reporter Assay System. Each experiment was done in triplicate and repeated at least two times. (3691)

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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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J. Biol. Chem. 282, 9883–94. Cell confluence-induced activation of signal transducer and activator of transcription-3 (Stat3) triggers epithelial dome formation via augmentation of sodium hydrogen exchanger-3 (NHE3) expression. 2007

Su, H.W., Yeh, H.H., Wang, S.W., Shen, M.R., Chen, T.L., Kiela, P.R., Ghishan, F.K. and Tang, M.J

Notes: The authors tested their hypothesis that Na+-H+ exchangers (NHE) are involved in the formation of multicullar dome structures in confluent Madin-Darby canine kidney (MDCK) cells and that the Stat3 pathway is involved in regulation of NHEs. The authors performed semi-quantitative RT-PCR to monitor NHE3 mRNA levels in MDCK cells expressing a constitutive Stat3 mutant or a dominant-negative Stat3 mutant. The reverse transcription step was performed using Promega M-MLV Reverse Transcriptase. RAlso, Stat3 activities in low-density cultures and high-density cultures were compared using a reporter gene assay. Four copies of the Stat3-binding site were cloned upstream of a firefly luciferase reporter gene, and the resulting vector, along with the pRL-TK Vector for normalization, were transfected into MDCK cells. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System. (3910)

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J. Biol. Chem. 282, 29847–29854. Differential regulation of vitamin D receptor (VDR) by the p53 family: p73-dependent induction of VDR upon DNA damage. 2007

Kommagani, R., Payal, V. and Kadakia, M.P.

Notes: The authors examined transcriptional regulation of the vitamin D receptor (VDR) by p53 and p63, a member of the p53 family, under stressed and unstressed conditions. Reporter constructs with the full-length and minimal VDR promoters controlling expression of firefly luciferase were cotransfected with p53 or p63 expression constructs, and transcriptional activation of the VDR promoter was monitored using the Dual-Luciferase® Reporter 100 Assay System. Results were normalized to Renilla luciferase activity. Interaction between p73, another member of the p53 family, and the VDR promoter was examined using chromatin immunoprecipitation. The imunnopreciptated chromatin was reverse crosslinked, DNA was eluted and VDR and p21 sequences were detected by PCR using GoTaq® Green Master Mix. (3715)

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J. Biol. Chem. 282, 10953–10962. Evidence for 1,25-dihydroxyvitamin D3-independent transactivation by the vitamin D receptor: uncoupling the receptor and ligand in keratinocytes. 2007

Ellison, T.I., Eckert, R.L. and MacDonald, P.N.

Notes: While the absence of the Vitamin D receptor (VDR) has profound effects in skin cells, mutation of 25-hydroxyvitamin D 1α-hydroxylase (24OHase), the enzyme required for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone biosynthesis, has little effect on the skin. To determine how VDR may transactivate independent of the 1,25(OH)2D3 ligand, the human 24-hydroxylase promoter was amplified from MCF-7 genomic DNA, digested with XhoI and HindIII and inserted into the pGL3-Basic Vector. Mutations in the proximal and distal vitamin D response elements in the human 24-hydroxylase promoter were introduced using the GeneEditor™ Site-Directed Mutagenesis System. HaCaT cells, primary human fibroblasts or primary human keratinocytes were seeded at a density of 3.2 × 104 cells/well in 12-well plates and transiently transfected with reporter constructs. After 18 hours, the cells were exposed to 1,25(OH)2D3, 9-cis-retinoic acid, ethanol vehicle, or no additive and harvested 24 hours later. The luciferase activity of the cell lysates was measured using the Dual-Luciferase® Reporter Assay System. Five micrograms of RNA purified from mouse keratinocyte and fibroblast cultures was reverse transcribed and amplified for the 24OHase transcripts using the PCR Master Mix. The products were analyzed on ethidium bromide-stained 2% agarose gels. (3695)

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J. Immunol. 178, 4517-4527. Evidence for evolving Toll-IL-1 receptor-containing adaptor molecule function in vertebrates. 2007

Sullivan, C., Postlethwait, J.H., Lage, C.R., Millard, P.J. and Kim, C.H.

Notes: The authors used reporter assays and coimmunoprecipitation experiments to compare the zebrafish (Danio rerio) Toll-IL-1R-containing adaptor molecule 1 (TICAM1) activation of NF-κB and zebrafish type I IFN to mammalian TICAM1 activation. 293H and ZFL (zebrafish liver) cells were cotransfected with 400 ng of TICAM1 construct (mouse, zebrafish or a deletion construct), 400 ng of a reporter construct (e.g., a zebrafish IFN promoter cloned into the pGL3-Basic Vector) and 10 ng of pRL-CMV Vector, an internal control to normalize data. After 24 hours for 293H cells and 48 hours for ZFL cells, the transfected cells were lysed, and luciferase activity was measured using the Dual-Luciferase® Reporter Assay System. For coimmunoprecipitation experiments, 293H cells were cotransfected with a total of 8µg of plasmids (3µg of zebrafish or mouse TICAM1 construct, 3µg of interacting protein construct, 1µg of the pAdVAntage™ Vector, and 1µg of antiapoptotic protein p35 construct). Forty-eight hours posttransfection, the cells were lysed, the protein bound to affinity resins and analyzed by Western blot. (3755)

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Clin. Vaccine Immunol. 14, 685-692. Fruit-specific expression of the human immunodeficiency virus type 1 Tat gene in tomato plants and its immunogenic potential in mice. 2007

Ramírez YJ, Tasciotti E, Gutierrez-Ortega A, Donayre Torres AJ, Olivera Flores MT, Giacca M, Gómez Lim MA.

Notes: In this study, the HIV Tat gene was expressed in tomato plants. Mice were given 10mg of tomato fruit extract from either transgenic or wild-type plants orally, intraperitoneally and intramuscularly. A strong anti-Tat immune response was obtained in mice immunized with the transgenic fruit, regardless of the administration route. Mice that received oral vaccination developed early evidence of mucosal immunity. Sera from the immunized mice inhibited Tat-dependent transactivation of the HIV long terminal repeat promoter in a luciferase reporter assay. (3706)

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J. Biol. Chem. 282, 37605–37617. Identification and characterization of a juvenile hormone response element and its binding proteins. 2007

Li, Y., Zhang, Z., Robinson, G.E. and Palli, S.R.

Notes: The authors characterized a juvenile hormone response element in Drosophila melanogaster (DmJHRE1) and identified two proteins that bound to a DmJHRE1 affinity column. Proteins eluted from the column were digested with Sequencing Grade Modified Trypsin, subjected to liquid chromatography-tandem mass spectrometry and identified as FKBP39 and Chd64. DmJHRE1 transcription regulatory activity was confirmed using reporter constructs with DmJHRE1 sequences regulating expression of firefly luciferase in Drosophila L57 and S2 cells. A vector with Renilla luciferase and the Autographa californica multicapsid nucleopolyhedrovirus IE1 promoter was used for normalization. Luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. Potential interactions between FKBP39, Chd64 and several candidates proteins for the JH receptor were examined using the MagneGST™ Pull-Down System. Each bait protein was expressed as a GST-fusion protein in E. coli and immobilized using MagneGST™ Glutathione Particles. [35S]Methionine-labeled prey proteins were expressed using the TNT® T7 Quick Coupled Transcription/Translation System. (3784)

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Cell 131, 861-872. Induction of pluriopotent stem cells from adult human fibroblasts by defined factors 2007

Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K. and Yamanaka, S.

Notes: The authors of this paper describe the generation of induced pluripotent stem (iPS) cells from human dermal fibroblasts. STR analysis using the PowerPlex® 16 System showed that patterns of 16 STRs in the clones matched the parent cell line. Luciferase assays to assess activity of the OCT3/4 and Rex1 promoters were performed using the Dual-Luciferase® Assay System. (3951)

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J. Biol. Chem. 282, 10290–10298. Interaction between sterol regulatory element-binding proteins and liver receptor homolog-1 reciprocally suppresses their transcriptional activities. 2007

Kanayama, T., Arito, M., So, K., Hachimura, S., Inoue, J. and Sato, R.

Notes: To explore the interaction of liver receptor homolog (LRH)-1, a known suppressor of sterol regulatory element-binding protein (SREBP) transcriptional activity, human LRH-1 was reverse transcribed then amplified by PCR from total RNA from HepG2 cells. The amplification product was ligated into the pTargeT™ Mammalian Expression Vector to create pTarget-LRH1. For reporter experiments, a PCR fragment that encompassed the 1.3kb 5’-promoter region of the human small heterodimer partner (SHP) gene was cloned into the pGL3-Basic Vector (designated pSRB). The pGL3-Promoter Vector was used to construct pLRHREx3, which contains three LRH-1 response elements, and the insert was generated using synthetic oligonucleotides. HEK293 cells were cotransfected with 0.2µg of a promoter-firefly luciferase construct, 0.1µg of a SREBP expression plasmid, 10ng of phRL-TK Vector and 0.2 or 0.6µg of pTarget-LRH1. Alternatively, the cotransfected plasmids were 0.2µg of pSHP, 0.1µg of pTarget-LRH1, 10ng of phRL-TK Vector and 0.2 or 0.6µg of a SREBP expression plasmid. The pLRHREx3 construct (0.2µg) was cotransfected with 0.1µg of a LRH-1 expression plasmid, 0.2µg of pCMXPGC-1α (peroxisome proliferator activated receptor γ coactivator-1α), 10ng of phRL-TK Vector, and 0.1 or 0.3µg of a pSREBP expression vector in HEK 293 cells. Luciferase expression was assayed 48 hours post-transfection using the Dual-Luciferase® Assay Reporter System. To express SREBPs and LRH-1 in vitro, inserts were ligated into the pTNT™ Vector, synthesized using the TNT® Coupled Transcription/Translation System with radiolabeled methionine. Ten microliters of the 35S-labelled protein was then used in a GST-pulldown assay. (3692)

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Nucl. Acids Res. 35, 2390–2402. Molecular mechanism of upregulation of survivin transcription by the AT-rich DNA-binding ligand, Hoechst33342: evidence for survivin involvement in drug resistance. 2007

Wu, J., Apontes, P., Song, L., Liang, P., Yang, L. and Li, F.

Notes: To study how Hoechst33342 upregulates the expression and promoter activity of survivin, a novel member of the inhibitor of apoptosis (IAP) protein family, nested deletions of the survivin promoter driving a firefly luciferase reporter gene (pLuc-1430c ) were created using the Erase-a-Base® System. The vector was digested with SalI, the ends filled in using α-phosphorothioate dNTPs, digested a second time with BamHI and subjected to Exonuclease III digestion at 25°C. Aliquots of the 5’ end deletions were removed every 15–30 seconds, religated, transformed and analyzed by PCR and sequencing. Transient transfection experiments were carried out using HeLa cells seeded in 24-well plates and cotransfected 490ng of a pLuc-survivin construct and 10ng of pRL-TK Vector or in U937 cells using 2µg of survivin promoter constructs. After 24 hours, the HeLa cells were treated with Hoechst33342 and harvested 8–24 hours later. For U937 cells, the medium was changed with or without added drugs and the cells lysed after 36 hours. Reporter expression was assessed using the Dual-Luciferase® Reporter Assay System. (3697)

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J. Biol. Chem. 282, 14194-14204. Regulation of the interleukin-7 receptor α-promoter by the Ets transcription factors PU.1 and GA-binding protein in developing B cells. 2007

Dekoter, R.P., Schweitzer, B.L., Kamath, M.B., Jones, D., Tagoh, H., Bonifer, C., Hildeman, D.A., and Huang, K.J.

Notes: The interleukin-7 receptor is composed of γ and α subunits, encoded by the genes il7rg and il7r, respectively. The α subunit is expressed in developing B cells and is downregulated upon maturation. These authors investigated the mechanisms of transcriptional regulation of the il7r gene using 5´ RACE, EMSA, RNA interference and chromatin immunoprecipitation analyses. Potential promoter regions identified by 5´ RACE analysis were cloned into the pGL3-Basic luciferase reporter vector for further study. The promoter constructs were transiently transfected into the 38B9 pro-B cell line along with the control pRL-TK Vector, which expresses Renilla luciferase, and the Dual-Luciferase® Reporter Assay System was used to assess luciferase activity from the various promoter constructs. The promoter construct having the highest activity was chosen, and site directed mutagenesis was used to identify specific regions within the promoter fragment that may be important for activity. Sequence analysis was then used to identify a conserved Ets transcription factor binding site within the putative il7r promoter region. To determine whether the ETS transcription factor GABP binds to this Ets region, the authors performed chromatin immunoprecipitation analysis with an anti-GABP antibody. Immunoprecipitated DNA was then PCR-amplified with primers specific for the Ets region or control primers. The Wizard® SV Gel and PCR Clean-Up System was used to purify the amplified fragments prior to semiquantitative PCR analysis. (3626)

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Mol. Cell. Biol. 26, 8448–8460. Specific isoforms of translation initiation factor 4GI show differences in translational activity. 2007

Coldwell, M.J. and Morley, S.J.

Notes: The authors explored the role of five different eukaryotic initiation factor (eIF) 4GI protein isoforms, which are encoded by alternatively spliced mRNAs, by using short interfering RNAs (siRNAs) to silence the eIF4GI gene. Three eIF4GI siRNA target sequences were evaluated for their ability to reduce eIF4GI mRNA levels in HeLa cells. To quantify the extent of gene silencing, a control plasmid that encodes an eIF4GI/Renilla luciferase fusion mRNA was created using the psiCHECK™-2 Vector. Cotransfection of HeLa cells with the eIF4GI siRNAs and psiCHECK™-2 control plasmid resulted in degradation of the eIF4GI/Renilla luciferase mRNA, leading to reduced Renilla luciferase activity and lower light output. The psiCHECK™-2 Vector encodes the firefly luciferase gene, which allowed normalization of Renilla luciferase expression. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. Quantitative PCR (qPCR) was used to quantify the silencing of endogenous eIF4GI mRNA splice variants. Prior to qPCR, total RNA was isolated from siRNA-expressing HeLa cells, then reverse transcribed using the ImProm-II™ Reverse Transcription System. qPCR was The pGEM®-T Easy Vector was used in the creation of plasmids encoding siRNA-resistant eIF4GI isoforms, which were transfected into siRNA-expressing HeLa cells to restore eIF4GI function. (3778)

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J. Biol. Chem. 281, 22656–22664. The SWI/SNF chromatin remodeling subunit BAF57 is a critical regulator of estrogen receptor function in breast cancer cells. 2007

Garcia-Pedrero, J.M, Kiskinis, E., Parker, M.G., and Belandia, B.

Notes: To examine the role that BAF57, a transcriptional modulator of the estrogen receptor (ER), may have in breast cancer, BT549 cells were transfected with a reporter vector (pGL3-Basic with two estrogen response elements and the human pS2 promoter), the control pRL-CMV Vector and combinations of the following expression vectors: mERα or hERβ, BAF57, SRC1e, SRC1a and RAC3. After 16 hours, the cells were treated with 17β-estradiol. Twenty-four hours later, the cells were harvested and the luciferase activities assayed using the Dual-Luciferase® Reporter Assay System. GST-BAF57 (full-length, N- or C-domain) fusion protein was bound to Sepharose beads and incubated with 17β-estradiol or vehicle and wildtype or one of various mERα interaction domain mutants, which have been expressed and labeled with 35S methionine using a TNT® rabbit reticulocyte lysate system. The beads were washed and analyzed for bound protein. ZR-75-1 cells were transfected with BAF57 siRNA then treated with 17β-estradiol 24 hours later. Cell proliferation was measured using the CellTiter® 96 AQueous One Solution Cell Proliferation Assay. (3599)

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