Pfr luc

Manufactured by Agilent Technologies

Sourced in United States

The PFR-Luc is a laboratory equipment product from Agilent Technologies. It is a tool designed for the detection and measurement of bioluminescent signals, specifically luciferase reporter assays. The core function of the PFR-Luc is to quantify the light output generated by luciferase-based reporter systems.

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Lab products found in correlation

Pfa2 creb, by Agilent Technologies (2 mentions) Caco 2 cell, by American Type Culture Collection (2 mentions) Mg132, by Merck Group (2 mentions) Sodium taurocholate hydrate, by Merck Group (2 mentions) Bapta am, by Cayman Chemical (2 mentions) Pm vector, by Takara Bio (2 mentions) 1 oleyl rac glycerol, by Merck Group (2 mentions) Celltiter 96 non radioactive cell proliferation assay, by Promega (2 mentions) Lovastatin, by AG Scientific (2 mentions) 1 2 3h n cholesterol, by PerkinElmer (2 mentions) Hek293, by American Type Culture Collection (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Psv β galactosidase, by Promega (1 mentions) Luciferase assay kit, by Agilent Technologies (1 mentions) Victor x3 multilabel plate reader, by PerkinElmer (1 mentions) 96 well half area white plate, by Corning (1 mentions) 24 well plate, by Greiner (1 mentions) Firefly luc assay reagent, by Nanolight Technology (1 mentions)

12 protocols using pfr luc

Luciferase Reporter Assay for MBD5

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Neuro-2a (N2A, ATCC# CCL-131), HEK-293 (ATCC# CRL-1573), and NIH-3T3 cells were maintained in Dulbecco's modified Eagle's medium (Cellgro, Mediatech, Inc) and supplemented with 10% fetal bovine serum (Gibco, Life Technologies), penicillin (100 U/ml), and streptomycin (100 μg/ml) (Gibco, Life Technologies) at 37°C with 5% CO₂ until 95% confluence was attained. GAL4-BD fusions with different versions of mouse MBD5 were co-transfected with the luciferase reporter plasmid pFR-Luc (Agilent Technologies). The vector pSV-β-Galactosidase (Promega Corporation) was also co-transfected for normalization purposes. After 48 h post-transfection, the cells were lysed and the luciferase activity was measured with Luciferase Assay kit (Agilent Technologies) according to manufacturer's instructions. Each experiment was repeated independently at least three times, and luciferase activity (Turner BioSystems 20/20n, Promega Corporation) and β-Galactosidase activity (microassay protocol, Agilent Technologies) were measured in duplicates.

Camarena V., Cao L., Abad C., Abrams A., Toledo Y., Araki K., Araki M., Walz K, & Young J.I. (2014). Disruption of Mbd5 in mice causes neuronal functional deficits and neurobehavioral abnormalities consistent with 2q23.1 microdeletion syndrome. EMBO Molecular Medicine, 6(8), 1003-1015.

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Light-Controlled Gene Expression Activation

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For light-controlled transcription activation, cells were cotransfected with the constructs pQP-T2A^{[11 (link)]} (Addgene #102583) and pFR-Luc (Agilent Technologies) in a 3:1 ratio in 24-well plates (Greiner Bio-One). Illumination of plates was applied directly in CO₂ incubator with 740/25 nm light (0.2 mW cm⁻²) in alternating cycles: 30 s light and 180 s darkness. To measure Fluc activity, transfected cells were lysed 48 h after transfection. Cells were washed with PBS, and then 100 μl of lysis buffer (20 mM Tris–HCl pH 8.0, 10% glycerol, 0.1% β-mercaptoethanol, 0.1% Triton X-100, 1 mM PMSF) was added to each well of 24-well plate and incubated on ice for 30 min. 10 μl of cell lysates were mixed with 20 μl of Firefly Luc Assay reagent (NanoLight Technology) in 96-well half-area white plates (Costar). Bioluminescence signal was measured using a Victor X3 multilabel plate reader (PerkinElmer).

Redchuk T.A., Karasev M.M., Omelina E.S, & Verkhusha V.V. (2018). Near-infrared light-controlled gene expression and protein targeting in neurons and non-neuronal cells. Chembiochem : a European journal of chemical biology, 19(12), 1334-1340.

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Overexpression and Mutagenesis of Fe65

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For overexpression studies, human Fe65 with a C-terminal FLAG-Myc tag was obtained from Origene (RC202003) and Fe65 sub-cloned into expression vectors with N-terminal GFP or FLAG tags (PS100048 and PS100014, respectively, Origene). Specific mutants of Fe65 were generated using the Quikchange II XL site directed mutagenesis kit (Agilent). Primer details can be provided on request. For luciferase reporter gene assays, pMST-APP [provided by Prof Kwok-Fai Lau (The Chinese University of Hong Kong), with the kind permission of Prof Thomas Südhof (Stanford University School of Medicine, USA)], pFR-Luc (Agilent) and pRL-TK (Promega) were used.

Langlands H., Blain P.G, & Jowsey P.A. (2016). Fe65 Is Phosphorylated on Ser289 after UV-Induced DNA Damage. PLoS ONE, 11(5), e0155056.

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CREB Transcription Activity Assay

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pGL3-PGC-1α promoter construct was a gift from Dr. Michael Shuen (York University, Canada). pFA2-CREB and pFR-Luc (Agilent, Santa Clara, CA) were used to measure CREB activity. Luciferase assays were performed as previously reported ^{22 (link)}

Babbar M., Huang Y., An J., Landas S.K, & Sheikh M.S. (2018). CHTM1, a novel metabolic marker deregulated in human malignancies. Oncogene, 37(15), 2052-2066.

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Transfection and Reporter Assay for SUMO

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Baby hamster kidney (BHK) cells were cultured in high-glucose Dulbecco's Modified Eagle's Medium with GlutaMAX (DMEM/GlutaMAX) supplemented with antibiotics/antimycotics and 10% foetal calf serum. They were transfected seeded at ~80% confluence in 24-well multi-dishes (Nunc, Denmark) using Lipofectamine 2000 (Invitrogen, Paisley, UK). Each plasmid (pCMVAD-Fpnsumo, pCMVAD-FpnFL, pM-SUMO Wt, and pM-SUMO Mt) was co-transfected with 50 ng pSVβgal (Promega, Southampton, UK) as internal control, and 50 ng of the GAL4 reporter gene pFR-Luc (Agilent). After 48 h, luciferase activity was determined using the luciferase assay reagent (Promega), and β-galactosidase (βgal) activity was determined with the Beta-Glo reagent (Promega). Luminescence measurements were taken in a microplate luminometer (Tropix TR717, Applied Biosystems, MA), and levels of luciferase activity were normalized with respect to βgal activity.

Bayele H.K, & Srai S.K. (2021). A disease-causing mutation K240E disrupts ferroportin trafficking by SUMO (ferroportin SUMOylation). Biochemistry and Biophysics Reports, 25, 100873.

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Evaluating PGC1α Transcriptional Activation

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PPRE × 3-TK-luc containing 3 × DR1 sites was a kind gift provided by Bruce Spiegelman (Addgene plasmid #1015) and was used as reporter^{25 (link)}. HepG2 cells (24-well plates) were transfected with reporter constructs using HilyMax reagent according to the manufacturer’s protocol^{23 (link)}. Luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). The relative luciferase activity was calculated by dividing the firefly luciferase activity with the Renilla luciferase activity.
To evaluate the transcriptional activation ability of PGC1α, we used GAL4-PGC1α, containing a full-length PGC1α fused to the GAL4 DNA binding domain, kindly provided by Bruce Spiegelman (Addgene plasmid #8892)^{26 (link)}. The luciferase assay was performed using GAL4-PGC1α and pFR-Luc (Agilent Technologies, Santa Clara, CA, USA).

Inoue C., Zhao C., Tsuduki Y., Udono M., Wang L., Nomura M, & Katakura Y. (2017). SMARCD1 regulates senescence-associated lipid accumulation in hepatocytes. NPJ Aging and Mechanisms of Disease, 3, 11.

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Investigating Lovastatin's Effects on Caco-2 Cells

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Caco-2 cells were obtained from American Type Culture Collection (Manassas, VA). Lovastatin was obtained from A.G. Scientific (San Diego, CA). Cell viability assay was conducted using the CellTiter 96® Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI). pM vector was purchased from Clontech Laboratories, Inc. (Mountain View, CA). The reporter plasmid pFR-Luc was obtained from Agilent Technologies (La Jolla, CA). SGA360 and SGA315 were synthesized as previously described ^{17 (link)}. Primary antibodies used for immunoblot analysis are listed in Supplementary Table 1. [1,2-³H(N)]-cholesterol was purchased from Perkin Elmer (Waltham, MA). Sodium taurocholate hydrate, 1-oleyl-rac-glycerol, and MG132 were obtained from Sigma Aldrich (St. Louis, MO). β-Naphthoflavone (BNF) was purchased from Indofine Chemical Company (Hillsborough, NJ). BAPTA/AM and AEBSF were purchased from Cayman Chemicals (Ann Arbor, MI). Lovastatin was purchased from A.G. Scientific (San Diego, CA).

Muku G.E., Kusnadi A., Kuzu G., Tanos R., Murray I.A., Gowda K., Amin S, & Perdew G.H. (2019). Selective Ah receptor modulators attenuate NPC1L1-mediated cholesterol uptake through repression of SREBP-2 transcriptional activity. Laboratory investigation; a journal of technical methods and pathology, 100(2), 250-264.

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Quantitative Receptor Activation Assay

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Cell extracts from transiently transfected 293T cells were generated by lysing cells in 2 × LDS buffer (Invitrogen) containing protease and phosphatase inhibitor tablets (Roche). Samples were then sonicated and used for western blot analysis by standard methods. Antibodies used for western blot analysis were generated in house. For luciferase assay, engineered HEK293 cells with CRISPR/Cas9-mediated FGFR1 gene deletion [30] (link) were transiently transfected with expression vectors encoding appropriate receptors under the CMV promoter, Renilla luciferase (pRL-SV40, Promega), GAL-ELK1 transcriptional activator fusion (pFA2-ELK1, Agilent), and a firefly luciferase reporter driven by GAL4 binding sites (pFR-luc, Agilent), using FuGENE HD Transfection Reagent (Promega). On the next day, the transfected cells were cultured for an additional 6–8 h in serum free DMEM-based media containing an appropriate agonist antibody at various concentrations. The cellular luciferase activity was determined using Dual-Glo Luciferase Assay System (Promega) and EnVision Multilabel Reader (PerkinElmer). Firefly luciferase activity was normalized to the co-expressed Renilla luciferase activity to calculate relative light unit and data presented as fold induction.

Chen M.Z., Chang J.C., Zavala-Solorio J., Kates L., Thai M., Ogasawara A., Bai X., Flanagan S., Nunez V., Phamluong K., Ziai J., Newman R., Warming S., Kolumam G, & Sonoda J. (2017). FGF21 mimetic antibody stimulates UCP1-independent brown fat thermogenesis via FGFR1/βKlotho complex in non-adipocytes. Molecular Metabolism, 6(11), 1454-1467.

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CREB Transcription Activity Assay

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Babbar M., Huang Y., An J., Landas S.K, & Sheikh M.S. (2018). CHTM1, a novel metabolic marker deregulated in human malignancies. Oncogene, 37(15), 2052-2066.

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Mammalian Expression Constructs for FE65 and GSK3β

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Mammalian expression constructs for wild-type FE65, myc-tagged FE65, GST-FE65 PTB2 (531–676) and HA-tagged GSK3β were as described^{32 (link)–35 (link)}. Phosphomimetic (T579E) and dephosphomimetic (T579A) mutants were generated by using QuikChange II site-directed mutagenesis kit (Agilent Technologies). The GFP-tagged constructs of FE65 PTB2 was generated by subcloning corresponding FE65 cDNA into pEGFP-N1, and T579 phosphomimetic and dephosphomimetic mutations were introduced by using QuikChange II site-directed mutagenesis kit. The pRcCMV GAL4-APP plasmid, which encodes for the chimeric APP695 with full-length GAL4 yeast transcription factor fused to C-terminus, was as described^{36 (link)}. GAL4-dependent firefly luciferase reporter pFR-Luc and Renilla luciferase reporter phRL-TK were from Agilent Technologies and Promega, respectively.

Lee Y.S., Chow W.N, & Lau K.F. (2017). Phosphorylation of FE65 at threonine 579 by GSK3β stimulates amyloid precursor protein processing. Scientific Reports, 7, 12456.

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