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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. Biol. Chem. 280, 19977-19985. A novel Myc-target gene, mimitin, that is involved in cell proliferation of esophageal squamous cell carcinoma. 2005

Tsuneoka, M., Teye, K., Arima, N., Soejima, M., Otera, H., Ohashi, K., Koga, Y., Fujita, H., Shirouzu, K., Kimura, H. and Koda, Y.

Notes: The authors used 5´ and 3´RACE to amplify the gene mimitin, and the resulting cDNA was cloned into the pGEM®-T Vector. A genomic DNA fragment containing the mimitin promoter sequence was amplified by PCR and cloned into the pGEM®-T Vector. The promoter was then cloned into the pGL3-Basic Vector. The activity of the wildtype and mutated promoters was determined using a luciferase assay. The pRL CMV Vector was used to normalize for differences in transfection efficiency. (3466)

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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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Cancer Res. 65, 10024–10031. Epigenetic regulation of WTH3 in primary and cultured drug-resistant breast cancer cells. 2005

Tian, K., Jurukovski, V., Wang, X-P., Kaplan, M.H. and Xu, H.

Notes: Methylation of the WTH3 promoter region has been associated with multidrug resistance in breast cancer cells in vitro. In this study, the effect of WTH3 in primary cultured multidrug resistant cells was evaluated, and the influence of a frequently methylated CpG23 region on WTH3 promoter activity was investigated using a luciferase reporter assay system. Promoter regions containing wildtype or mutated CpG regions were cloned into the pGL3 Vector and transfected into paired MCF7 cell lines, along with a control pGL3 Vector without insert, and a plasmid containing the β-galactosidase gene as a transfection efficiency control. Luciferase activity was measured using the Steady-Glo® Luciferase Assay System, and β-galactosidase activity was determined using the Beta-Glo® Assay System. Luciferase activities of the wildtype and mutant promoter regions were compared after normalization to β-galactosidase activity and protein concentration. (3457)

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J. Biol. Chem. 278 (52), 52739-52746. Expression of a human surfactant protein C mutation associated with interstitial lung disease disrupts lung development in transgenic mice.  2005

Bridges, J.P., Wert, S.E., Nogee, L.M. and Weaver, T.E.

Notes: A minimal promoter from the gene encoding BiP was cloned into the pGL3-Basic Vector. The resulting construct was transfected into HEK293 cells and, 48 hours post transfection, cell lysates were prepared using Glo Lysis Buffer. The Bright-Glo™ Luciferase Assay System was then used to assess levels of luciferase expression.  As a transfection control, cells were co-transfected with the pSV-β-Galactosidase Control Vector.  (3229)

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Nucl. Acids Res. 33, 4140–56. Functional polarity is introduced by Dicer processing of short substrate RNAs. 2005

Rose, S.D., Kim, D.H., Amarzguioui, M., Heidel, J.D., Collingwood, M.A., Davis, M.E., Rossi, J.J. and Behlke, M.A.

Notes: Having observed that blunt 27mers had increased potency in RNAi compared to 21mers or 27mers with 3' or 5' overhangs, these authors investigated what differences may account for these changes in gene silencing activity using the same target sequence in enhanced green fluorescent protein (EGFP). For one experiment, a PCR-generated fragment of the EGFP coding region spanning sites EGFPS1 and EGFPS2 was cloned into psiCHECK™-2 Vector in both sense and antisense orientations. Also, a PCR-generated fragment of the human heterogeneous nuclear ribonucleoprotein H (hnRNPH) coding region spanning sites H1 and H3 was similarly cloned in sense and antisense orientations. HEK293 cells were transfected with 150ng EGFP sense and antisense vectors plus EGFPS2 or control duplex RNAs. HCT116 cells were transfected with 100ng sense and antisense hnRNPH vectors with H3 or control duplex RNAs. The Dual-Luciferase® assay was used to evaluate luciferase expression 24 hours post-transfection. In a separate EGFP RNAi experiment, the Steady-Glo® Luciferase Assay System was used to monitor firefly luciferase activity to normalize transfection of HEK 293 cells. A further RNAi experiment targeted the firefly luciferase gene in the pGL3-Control Vector cotransfected with 20, 2 or 0.4 nM siRNA duplexes into HeLa cells. After 48 hours, the cells were lysed and 10µl tested using the Luciferase Assay System. To test the level of expression of human La antigen targeted for gene silencing, total RNA was harvested from HeLa cells using the SV 96 Total RNA Isolation System, reverse transcribed and used in real-time PCR. (3289)

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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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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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Proc. Natl. Acad. Sci. USA 101(9), 2794-2799. Differential utilization of upstream AUGs in the beta-secretase mRNA suggests that a shunting mechanism regulates translation. 2004

Rogers G.W. Jr, Edelman G.M. and Mauro V.P.

Notes: The authors studied the behavior of the three upstream AUGs to the initiation codon of beta-Secretase (BACE1) in a cell-free expression system to understand how this 5´ upstream region affects translation.  The 5´ UTR (with or without a hairpin structure) was cloned in front of the firefly luciferase gene derived from the pGL3-Control Vector. The resultant construct was either transfected into cells or in vitro transcribed to yield capped or uncapped RNA followed by translation in Rabbit Reticulocyte Lysate, Nuclease Treated, with or without m7GpppG cap analogue.  The experiment demonstrated that both the hairpin structure and the 5´ cap analogue decreased the translation level of the BACE1 protein.  (3076)

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J. Immunol. 172, 5512–5521. Human CD1D gene has TATA boxless dual promoters: An SP1-binding element determines the function of the proximal promoter. 2004

Chen, Q.Y. and Jackson, N.

Notes: These authors demonstrated that the human CD1D gene has distal and proximal TATA boxless promoter sequences. Distal and proximal promoters to CD1D were cloned into the pGL3-Basic Vector to create reporter constructs. One construct contained the entire 4,986 base pair region containing distal and proximal CD1D promoter.  Transient transfections were performed using 5 x 105 Jurkat cells in 24-well plates, 0.8 μg of pGL3 Basic Vector with the insert of interest, and 30ng of pRL-CMV Vector as a transfection normalization control. The Dual-Glo™ Luciferase Assay System was used to assay luciferase activities.  (3061)

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Cancer Res. 64, 7473-7478. Human polynucleotide phosphorylase (hPNPase0ld-35): A potential link between aging and inflammation 2004

Sarkar, D., Lebedeva, I.V., Emdad, L., Kang, D-c., Baldwin, A.S. and Fisher, P.B.

Notes: Human polynucleotide phosphorylase (hPNPaseold-35) was originally identified as a gene that is upregulated during cellular differentiation and senescence. The authors of this study show that overexpression of hPNPaseold-35 results in the increased production reactive oxygen species (ROS) and the activation of the NF-κB pathway, resulting in expression of the inflammatory cytokines IL-6 and IL-8. HeLa cells were transfected with either empty pGL3-Basic or 3kB-Luc (pGL3-Basic containing three tandem NF-κB binding sites). Transfected cells were infected with an empty adenoviral vector or one containing hPNPaseold-35. Luciferase assays were conducted using the Luciferase Assay System, and signal was normalized to a pSV-βgal control. A 10- to 12-fold increase in luciferase activity was observed in the cells infected with the adenovirus containing hPNPaseold-35 compared to the control cells. This activation was inhibited by compounds that reduced ROS. The authors suggest that hPNPaseold-35 plays an important role in producing age-related inflammation. (3643)

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Am. J. Physiol. Cell Physiol. 287, 1031-1040. Molecular analysis of fiber type-specific expression of murine myostatin promoter. 2004

Salerno, M.S., Thomas, M., Forbes, D., Watson, T., Kambadur, R., and Sharma, M.

Notes: In this article, a 2.5- and a 1.7-kb fragment of the myostatin promoter were amplified by PCR and cloned into the pGEM®-T Easy vector.  These clones were then subcloned into the pGL3-Basic Vector.  Deletion mutants of the constructs were made and used to transient transfect mouse muscle C2C12 cells.  The Luciferase Assay System was then used to analyze transfectants.  The researchers also injected luciferase-reporter constructs into mouse muscle.  Luciferase activity in the mouse muscle was examined by grinding isolated muscles in liquid nitrogen and resuspending them in Cell Culture Lysis Buffer.  Ten micron cryosections of the muscles were also used in immunohistochmical staining experiments.  For these experiments, the researchers used a 1:50 dilution of Promega’s polyclonal anti-luciferase antibody, a secondary antibody and tertiary fluorescein-labeled conjugate.  The slides were counter stained with DAPI. (3147)

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J. Cell Biol. 279, 50069–50077. p51/p63 controls subunit 3 of the major epidermis integrin anchoring the stem cells to the niche. 2004

Kurata, S., Okuyama, T., Osada, M., Watanabe, T., Tomimori, Y., Sato, S., Iwai, A., Tsuji, T., Ikawa, Y. and Katoh, I.

Notes: HeLa and Saos-2 were transiently transfected with luciferase reporter vectors made from the pGL3-Promoter vector containing various intron sequences from the human integrinα3 gene ITGA3. The first intron sequence was originally cloned by PCR cloning into the pGEM®-T Easy vector. Cell cultures were assessed for luciferase activity by making lysates with the Glo Lysis Buffer and assaying with the Steady-Glo® Assay kit. (3225)

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Plant Cell 16(5), 1235-1250. Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing. 2004

Dunoyer P., Lecellier C.H., Parizotto E.A., Himber C. and Voinnet O.

Notes: The authors cloned five distinct silencing suppressor proteins from five different plant viruses in order to examine the pathways involving both small interfering RNA and micro RNA in Arabidopsis thaliana. These viral factors [P1- HcPro of Turnip mosaic virus (TuMV), P38 protein of Turnip crinkle virus (TCV), P19 protein of Tomato bushy stunt virus(TBSV), P25 protein of Potato virus X, and the P15 protein of Peanut clump virus (PCV)] were inserted into a mammalian expression vector and tested for protein production using the TNT® Quick Coupled Transcription/Translation System. The suppression effects of these plant viral proteins were also tested in HeLa cells. The CMV promoter was cloned from pRL-CMV into the pGL3-Basic Vector and both plasmids were transfected at 500ng each plus 1µg of each of the five suppressor-expressing vectors. After one day, 300ng siRNA targeting the firefly luciferase gene was added. Twenty-four hours later, the Dual Luciferase® Reporter Assay System was used to determine the ratio of firefly:Renilla luciferase expression and see if the viral suppressor protein had an effect. (3085)

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Mol. Cell. Biol. 24(8), 3337-46. Recruitment of N-CoR/SMRT-TBLR1 corepressor complex by unliganded thyroid hormone receptor for gene repression during frog development. 2004

Tomita A., Buchholz D.R. and Shi Y.B.

Notes: The authors used Xenopus laevis oocytes to show that unliganded thyroid hormone receptor (TR) recruits N-CoR (nuclear receptor corepressor) to modulate metamorphosis.  To study this influence, the cytoplasm of stage VI oocytes from X. laevis was injected with the indicated mRNAs [TR, retinoic acid receptor (RXR) and FLAG-tagged N-CoR].  The reporter plasmid TRE-Luc (0.33 ng/oocyte; thyroid hormone response elements from a Xenopus promoter driving expression of the firefly luciferase gene) and the control vector phRG-TK (0.03 ng/oocyte) were co-injected into the germinal vesicle (nucleus) after mRNA injection. After overnight incubation at 18°C, oocyte lysates were prepared by lysing six oocytes in 90µL 1X Passive Lysis Buffer. The Dual-Luciferase® Reporter Assay System was then used to assay 7µl of the lysate. The researchers also used an expression vector (based on the pGEM®-4Z Vector) containing the 5’ and 3’ untranslated regions of the X. laevis beta-globin gene flanking the multiple cloning site. (3088)

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Hum. Mol. Genet. 13, 2221-2231. SNPs in the promoter of a B cell-specific antisense transcript, SAS-ZFAT, determine susceptibility to autoimmune thyroid disease. 2004

Shirasawa, S., Harada, H., Furugaki, K., Akamizu, T., Ishikawa, N., Ito, K., Ito, K., Tamai, H., Kuma, K., Kubota, S., Hiratani, H., Tsuchiya, T., Baba, I., Ishikawa, M., Tanaka, M., Sakai, K., Aoki, M., Yamamoto, K. and Sasazuki, T.

Notes: Real-time TaqMan® amplification reactions were cleaned up using the Wizard® MagneSil® Sequencing Reaction Clean-Up System.  The purified products were then used in sequencing reactions.  This paper also describes use of the Dual-Luciferase® Reporter Assay System to analyze HEK293 cells transfected with a pGL3-Enhancer vector construct.  (3181)

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Mol. Cancer Res. 1, 475-84. Gene expression profiling in prostate cancer cells with Akt activation reveals Fra-1 as an Akt-inducible gene. 2003

Tiwari, G., Sakaue, H., Pollack, J.R., and Roth, R.A.

Notes: These authors analyzed gene expression profiles in the prostate cancer cell line PC3 upon induction of Akt activity to try to identify genes regulated by Akt that participate in the transformation of cells. They identified one mRNA of interest (Fra-1) and cloned its 5' regulatory region into a pGL3-Basic firefly luciferase reporter construct. This construct was used to transiently transfect MCF7 human breast cancer cells along with an Akt plasmid construct and a control vector expressing Renilla luciferase. The firefly construct was induced 4- to 5-fold by co-transfection with Akt3. Transfection conditions were as follows: MCF-7 cells were grown to 70% confluence in six-well plates, then incubated for 15 min with a mixture of 5ng of the control Renilla plasmid, 0.5μg of the Akt-expressing plasmid, and 0.5μg of pFra-luc construct and Fugene® 6 reagent at a 3:1 transfection reagent:DNA ratio. After 48 hours, luciferase activity was assessed using the Dual-Luciferase® Reporter Assay System.


(4361)

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J. Biol. Chem. 278 (22), 19691-19701. Complex transcription and splicing of odorant receptor genes. 2003

Volz, A., Ehlers, A., Younger, R., Forbes, S., Trowsdale, J., Schnorr, D., Beck, S. and Ziegler, A.

Notes: Total RNA was isolated from human testes using the RNAgents® Total RNA Isolation System. The isolated RNA was used in reverse transcription reactions to make cDNAs of HLA-linked OR genes. Promoter regions of HLA-linked OR genes were cloned into the pGL3-Basic Vector. The constructs were then transfected into human embryonic kidney (HEK293) and Odora cells for promoter analysis studies. The Bright-Glo™ Luciferase Assay System was used to generate data from the study.  Results were presented as a comparison to pGL3-Control Vector transfectants. (2730)

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J. Gene Med. 5(8), 723-732. Development of a dual-luciferase fusion gene as a sensitive marker for site-directed DNA repair strategies 2003

Bennett, M. and Schaack, J.

Notes: These authors created a construct encoding a Renilla and firefly luciferase fusion protein to examine the efficiency of site-specific DNA repair. The Renilla gene was taken from the pRL-null Vector and the firefly luciferase gene originated from the pSP-luc+NF Fusion Vector. The fusion construct was created by ligating the C-terminus of the Renilla luciferase gene to the N-terminus of the firefly gene.  The fused proteins were expressed at a constant ratio when transfected into mammalian cells.  Firefly luciferase expression was eliminated by deleting a T at position 213 creating an ochre translational stop codon and placing the downstream sequence out of frame. The mutant protein was then tested for repair using two methods: small fragment homologous replacement and oligonucleotide-mediated repair. The Renilla:firefly expression ratio was tested in several human, murine and simian cell lines and assayed 48 hours post-transfection using the Dual-Luciferase® Reporter Assay System.  In addition, the repaired plasmids were recovered to verify the sequence correction. (3064)

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Cancer Res. 63, 1818-1821. Firefly luciferin-activated Rose Bengal: In vitro photodynamic therapy by
intracellular chemiluminescence in transgenic NIH 3T3 cells.
2003

Theodossiou, T., Hothersall, J.S., Woods, E.A., Okkenhaug, K., Jacobson, J. and MacRobert, A.J.

Notes: NIH 3T3 cells were transfected using Superfect (Qiagen) with Promega's pGL3-Control Vector and a marker for antibiotic resistance at a 5:1 ratio. Transfected cells were visualized after in situ addition of 500μM Beetle Luciferin in 10% FCS medium.  The transfected cells were used to show that light from a luciferase reaction can be used to create targeted cytotoxicity in the presence of the photosensitizer Rose Bengal (RB). This application has specific relevance as a  tumor treatment by photodynamic therapy. (2671)

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Clin. Can. Res. 9, 3167-3175. Fully human anti-interleukin 8 antibody inhibits tumor growth in orthotopic bladder cancer xenografts via down-regulation of matrix metalloproteases and nuclear factor-κB 2003

Mian, B.M., Dinney, C.P.N., Bermejo, C.E., Sweeny, P., Tellez, C., Yang, X.D., Gudas, J.M., McConkey, D.J., Bar-Eli, M.

Notes: Luciferase reporter gene constructs based on the pGL3 vector that contained either MMP-2 or MMP-9 promoters, SV40 promoter (positive control ) or the luciferase basic vectors were transfected into metastatic cells, S53JB-V cells or UMUC-3 cells along with a pβ-actin RL control construct. Cells were treated with an anti-IL-8 antibody, IgG control or no antibody. Dual luciferase reporter assays were performed to determine the effect of the treatments on activity and expression of the MMP-2 and MMP-9 constructs. (2716)

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FEBS Lett. 555, 390-396. Hsp105 but not Hsp70 family proteins suppress the aggregation of heat-denatured protein in the presence of ADP. 2003

Yamagishi, N., Ishihara, K., Saito, Y., and Hatayama, T.

Notes: The pGL3-Control Vector was co-transfected into COS-7 cells with mammalian expression vectors expressing either heat shock protein Hsp70 or Hsp105α.  The cells were ATP-depleted in glucose-free media with 2μM rotenone and 5mM 2-deoxyglucose for up to 3 hours. Afterwards the cells were lysed with the Cell Culture Lysis Reagent and assayed for luciferase activity uaing the Luciferase Assay System. ATP depletion was verified during 2μM rotenone and 5mM 2-deoxyglucose treatment using the CellTiter-Glo® Assay.  (2841)

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J. Virol. 77, 4588-4596. Human Cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like Receptor 2. 2003

Compton, T., Kurt-Jones, E.A., Boehme, K.W., Belko, J., Latz, E., Golenbock, D.T., and Finberg, R.W.

Notes: Researchers used Promega's pGL3-Basic Vector to create a NF-κB promoter-driven firefly luciferase reporter vector for use in transient transfections with the phRL-TK Vector. The two vectors, along with pUC18, were transiently transfected into HEK293 cells using GeneJuice Transfection Reagent (Novagen). Transfections were performed overnight using 80ng NF-κB-pGL3-Basic construct, and 20ng of phRL-TK and pUC18 vectors in 96-well plates containing 2.5 x 104 cells/well. The following day, the cells were exposed to various treatments. Luciferase activity was measured using the Dual-Glo™ Luciferase Assay System. (2672)

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J. Biol. Chem. 278, 9332-9338. Identification of an upstream enhancer in the mouse laminin α1 gene defining its high level of expression in parietal endoderm cells. 2003

Niimi, T., Hayashi, Y. and Sekiguchi, K.

Notes: The pGL3-basic and pGL3-Promoter vectors were used to make constructs with varying lengths of the mouse LAMA1 promoter. Gene fragments up to 6.1 kb were cloned into the pGL3-promoter vector and analyzed for luciferase expression in a variety of cell lines using the Dual-Luciferase® Reporter Assay System. F9, NIH/3T3, PYS-2 and EHS cells were transiently transfected with 200ng of reporter construct and 20ng of the Renilla luciferase-expressing phRL-null vector in 24-well plates.  Forty-eight hours later, cell lysates were harvested with Passive Lysis Buffer. Site-directed mutagenesis of  the LAMA1 gene promoter was performed with the GeneEditor™ in vitro Site-Directed Mutagenesis System.  (2631)

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