Notes: Researchers created promoter constructs in the pGL3-Basic vector to study NHE3 promoter function in cotransfection experiments with the pRL-null vector.  Transfected Caco-2 cells were analyzed by the Dual-Luciferase^® Reporter Assay System. Beta-Galactosidase Assays were also performed on SL2 cells cotransfected with the pRL-null vector. The Renilla Luciferase Assay System was used to normalize these transfectants. (2642)

Notes: The Y-box-binding protein YB-1 was examined through the use of a reporter plasmid with wildtype and mutant putative Y-box binding sites. The binding sites were constructed in the pGL3 Basic Vector and measured using the Luciferase Assay System. Studies were performed in normal human dermal fibroblasts.  Expression of YB-1 was found to repress expression of the COL1A2 gene at the transcriptional level. Confirmation of this dose-dependent inhibition was through measurement of the steady-state mRNA levels by quantitative, real-time RT-PCR.  The reverse transcription portion of the quantitative, real-time RT-PCR reactions were performed with ImProm-II™ Reverse Transcriptase followed by a TaqMan-type quantitative PCR step. (2623)

Notes: Drosophila Mi-2 was discovered to specifically bind the DNA binding domain of dDREF (DNA replication-related element factor) by yeast two hybrid screen. This binding was shown to inhibit dDREF’s transcriptional regulatory action on a DNA replication-related element (DRE). Analysis of dDREF binding to the DRE was performed by examination of expression from –168DPCNAluc, –168mutΔPCNAluc, and pRL-actin5C reporter genes in Schneider cells using the Dual-Glo™ Luciferase Assay System. (2622)

Notes: This study investigated the effect of aberrant expression of EAAT2 mRNA on expression of the EAAT2 protein in human glioma cells. EAAT2 encodes an excitatory amino acid transporter that helps to clear glutamate from synaptic cleft. In transfection-based studies on human U251 gliomal cells,  transient transfection efficiency was evaluated using a luciferase reporter gene, and luciferase activity was detected with the Luciferase Assay System. In separate experiments, RTPCR products were cloned into plasmid vectors. Plasmid DNAs containing PCR products were isolated from bacteria using the Wizard^® Plus SV Minipreps DNA Purification System. (2607)

Notes: An IL-1 inducible fragment of the murine IL-2 promoter and five tandemly-arranged NFκB binding sites were cloned into the pGL3-Basic Vector, creating the constructs pGL3-IL-2 and pGL3-5xNFκB, respectively. IL2 promoter activation and NFκB activation were measured after rhIL-1α - or PMA-stimulation of EL4D6/76 cells transiently transfected with these constructs. Luciferase activity was measured using the Steady-Glo® Luciferase Assay System. As an internal control, cells were co-transfected with the pRL-SV40 Vector, and expression of firefly and Renilla luciferase was measured using the Dual-Luciferase® Reporter Assay System. (2427)

Notes: To investigate the effect of the compound kamebakaurin (KA) on NF-κB, an NF-κB-responsive firefly luciferase vector was transfected into HeLa, Jurkat and THP-1 cells. The Luciferase Assay System was used to assay the level of NF-κB induction after treatment of cells with various concentrations of KA. To determine if KA influenced the DNA-binding activity of NF-κB, nuclear extracts of HeLa, Jurkat and THP-1 cells were prepared after preincubation with KA and stimulation of NF-κB activity. Control nuclear extracts were prepared from unstimulated p50- or RelA-overexpressed MCF-7 cells. In addition, the wildtype and DNA-binding mutant RelA and p50 (NF-κB) His-tagged proteins were translated using the TNT^® Quick Coupled Transcription/Translation System and subsequently purified. Using the Gel Shift Assay System, the NF-κB and AP1 oligos were tested for electromobility shifts with the prepared nuclear extracts or with purified wildtype and mutant proteins. Supershift studies using anti-p50 or anti-RelA antibodies were also performed. (3113)

Notes: These authors used the Steady-Glo^® Luciferase Assay System and Reporter Lysis Buffer in their studies on the 5´ UTR of p27 mRNA. (2434)

Notes: In this study, the Steady-Glo^® Luciferase Assay System was used to monitor luciferase activity in COS-1 and HeLa cells transfected with various reporter constructs. (2436)

Notes: The authors of this study have developed a high throughput method for screening protein-protein interactions in 384-well plates. Using elements of the CheckMate™ Mammalian Two-Hybrid System, mouse cDNAs were linked to the GAL4 DNA binding and VP16 transcription activation domains via a linear cassette PCR strategy. The linear DNAs were mixed with the pG5luc plasmid, transfection reagent and CHO-K1 cells.  Twenty hours after transfection, activation of luciferase by GAL4 binding was assayed using the Steady-Glo^® Luciferase Assay System. Biotec and Tecan liquid handlers were used to semiautomate the system, allowing screening of 20,000 assay wells per day. (2727)

Notes: To study the transcriptional control of human CYP8B1 by bile acids, the -514/+300 region of the gene was cloned into the pGL3-Basic Vector and subsequently mutagenized at both the 5´ and 3´ends of the CYP8B1 fragment.  The various reporter constructs were then transfected into HepG2 cells and the activity assessed using the Luciferase Assay System. Luminescence was determined using a Berthold Lumat LB 9501 luminometer. The Gal4/Id and VP16/MyoD provided in the CheckMate™ Mammalian Two-Hybrid System were used as positive controls for the in a two-hybrid assay that examined the interaction of SHP and HNF4alpha, genes that regulate CYP8B1.  The TNT^® Quick Coupled Transcription/Translation System was used to synthesize various receptor proteins cloned in expression plasmids. (3083)

Notes: The pTARGET™ Mammalian Expression Vector was used to clone and express a portion of the ARA9, a protein that interacts with the aryl hydrocarbon receptor. The expressed protein was used in a two-hybrid assay using a GAL4-fusion protein containing a portion of the aryl hydrocarbon receptor. Interaction of the two proteins is reported by activation of the luciferase in the pG5-luc Vector, which is a portion of the CheckMate™ Mammalian Two-Hybrid System. The two-hybrid studies were performed in COS-1 cells and were controlled with a cotransfection with a β-galactosidase vector. Cells were lysed with the Passive Lysis Buffer and luciferase activities determined with the Luciferase Assay System. (0842)

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

Notes: In this study, a fragment of the nitric oxide synthase type 2 (NOS2) promoter was cloned into the pGL3-Basic Vector. C6 glioma cells stably transfected with this construct were then exposed to various NOS2 inducers and lactacystin. Luciferase activity was measured using the Steady-Glo^® Luciferase Assay System. (2435)

Notes: The interleukin (IL)-1ß stimulated release of granulocyte macrophage colony stimulating factor (GM-CSF) from lung epithelial cells was explored in this study. To test the promoter activity of GM-CSF, the promoter and enhancer regions were amplified by PCR from human peripheral blood mononuclear cells and cloned into the pGEM^®-T vector. After verification by sequencing, the promoter and enhancer were cloned individually and together into the pGL3-Basic Vector. Additionally, an Xho I/Sal I fragment containing the HSV tk promoter, a gene conferring neomycin resistance, and a poly-A tail were cloned into the Sal I site of pGL3 to allow the production of stable transfectants. To perform stable transfections, 20µl of the Tfx™-50 transfection reagent was incubated with 8µg of plasmid in serum-free medium for 15 minutes at room temperature.  Preconfluent human A549 type II alveolar carcinoma cells were incubated with the transfection mix for 2 hours after washing with serum-free medium. The cells were then cultured in fresh medium for 16 hours before the addition of  0.5mg/mL G-418. After 21 days, foci of cells developed and were harvested for use in luciferase assays. The cells were plated into 24-well plates, grown to confluency and incubated in serum-free medium. Stimulation by 1ng/ml IL-1ß or 1µM PMA for 12 hours was followed by lysate production and measurement of luciferase activity using the Luciferase Assay System and a Turner luminometer. Luciferase readings were standardized against total protein measurements. The PolyATract^® System IV was also used prior to a Northern Blot to obtain purified mRNA. (2742)

Notes: The authors identified c-E10, a protein that has an N-terminal caspase-recruiting domain, in a screen for molecules involved in regulation of death receptor signaling pathways. A cDNA encoding viral-E10 was transcribed and translated in vitro using Promega TNT Coupled Reticulocyte Lysate Systems. Additionally, HeLa cells were transfected with a variety of cDNAs together with a β-galactosidase reporter gene, treated with TNFα, and then assayed for cell death using the CellTiter^® Non-Radioactive Cell Proliferation Assay. Expression of c-E10 did not affect the ability of TNFα to induce apoptosis in the transfected cells.  The authors also used the Luciferase Assay System to assess NF-κB activation in the transfected cells. (2502)

Notes: Treatment of RAW 264.7 cells were treated with CpG-oligonucleotides derived from bacterial DNA. The cells responded by releasing TNFα and IL-12. The MEK Inhibitor U0126 inhibited the release of the proteins. The inhibitor has no effect on the TNFα promoter or mRNA levels but has a big effect on IL-12 promoter activity and mRNA levels, suggesting different mechanisms of MAPK pathway involvement. Promoter studies were performed with constructs made in the pGL3-Basic Vector and analyzed with the Luciferase Assay System. (1091)

Notes: IEC-6 cells were seeded into 96-well plates and cultured in the presence of various concentrations of human IL-17. Cell proliferation was measured using the CellTiter 96^® AQ_ueous Non-Radioactive Cell Proliferation Assay. The pGL3 vector was used to clone a PCR-amplified piece of the 5' flanking region of the CINC gene, and the Luciferase Assay System was used to assess promoter activation. All transfections of IEC-6 cells used the pSV-β-galactosidase vector as a control for transfection efficiency. Total RNA for Northern blotting was isolated using the Poly ATtract System. (2510)

Notes: The authors use the Luciferase Assay System to measure luciferase activity in mammalian and insect cells 40-48 hours after transfection. The TNT^® T7 Coupled Reticulocyte Lysate System is used to generate in vitro translated and [³⁵S]methionine-labeled proteins for in vitro protein-protein interaction assays (2412)

Notes: The authors used the Dual-Luciferase^® Reporter Assay System, pGL3 Control Vector, pRL-CMV Vector, pSV-beta-Galactosidase Control Vector, Luciferase Assay System and RNase ONE^® Ribonuclease in their studies. The objective of the paper was to demonstrate that the 5' untranslated region (UTR) of c-myc functions as an internal ribosome entry site (IRES). To directly assay the ability of the UTR to function as a IRES, the Renilla luciferase gene was subcloned in front of the UTR. Dual expression of the luciferases was demonstrated in both HeLa cells and HepG2 cells and dependent upon the c-myc UTR. Mutation of the Renilla luciferase sequence to contain a hairpin structure, demonstrate that the Renilla luciferase activity could reduced by 75% with no effect on the firefly luciferase activity. RNase protection assays with a 624 nucleotide probe that crossed the Renilla luciferase cDNA, the c-myc UTR and the firefly luciferase cDNA demonstrated that only one single message is produced by the transfected HeLa cells and the c-myc UTR is not functioning as a promoter. All transfections were normalized to control β-galactosidase activity. (0339)

Notes: Either wildtype or mutant Mv1Lu cells were transfected using Tfx™-50. No details are provided. Luciferase activity in the cell lysate was measured the following day using Promega's Luciferase Assay System. (0584)

Notes: IL-1β and TNFα  have been shown to induce two p50/p65 NF-κB DNA binding complexes in A549 cells. Tfx™-50 Reagent was used to stably transfect A549 cells in order to study the effects on NF-κB-dependent transcription. Promega's Luciferase Assay System was used to measure the effect of kinase inhibitors on IL-1β and TNFα stimulated NF-κB-dependent transcription. In addition, Promega's NF-κB and OCT1 Oligonucleotides were used in gel shift assays (1438)

Notes: The ProFection® Mammalian Transfection System - Calcium Phosphate was used to transfect HeLa and HeLa-Tat cell lines. The authors developed a vector-antiviral gene system for use in gene therapy in the treatment of HIV-1. pGEM®-luc was used as a negative control in luciferase assays of transfected cells. (1388)

Notes: A reporter construct was assembled in the pGL3 Control Vector downstream of the Xba I consisting of the 3'UTR of the COX-2 gene. The construct was designed to see if the UTR had an affect on mRNA stability in the presence of IL-1beta. The construct as well as the pSV-Beta Galactosidase Control Vector were transfected into A549 cells using the Tfx™-50 Reagent. A lot of detail is provided for the transfection. The reporter activities were monitored with the Luciferase Assay System and the Beta-Galactosidase Enzyme Assay System. (0606)

Notes: DNA-liposomes were injected intravenously with a RSV-luciferase expression vector. Forty-eight hours post injection, the animal was sacrificed and the heart, lung and ~300mg of liver was homogenized in Promega's Luciferase Cell Culture Lysis Reagent with the aid of zirconium beads. The debris was centrifuged away and the supernatant analyzed for luciferase activity with the Luciferase Assay System. (0199)

Notes: The 293 cell line was cotransfected with a luciferase reporter containing the IL-2 promoter, an expression vector for either EGR-1, NFATc or both and finally the pRL-TK Vector. Reporter to control ratio was 4:1 and the luciferase activity was determined with the Dual-Luciferase® Reporter Assay System. (1265)