Prl tk vector

Manufactured by Promega

Sourced in United States, China, United Kingdom, Switzerland

The PRL-TK vector is a plasmid used as a reporter gene in cell-based assays. It contains the Renilla luciferase (Rluc) gene, which serves as a reporter for gene expression and transcriptional activity. The vector also includes the Herpes Simplex Virus thymidine kinase (HSV-TK) gene, which can be used for selection purposes.

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Lab products found in correlation

Dual luciferase reporter assay system, by Promega (126 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (92 mentions) Pgl3 basic vector, by Promega (31 mentions) Dual glo luciferase assay system, by Promega (22 mentions) Dual luciferase assay system, by Promega (20 mentions) Dual luciferase reporter assay kit, by Promega (18 mentions) Dual luciferase reporter assay, by Promega (17 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (16 mentions) Passive lysis buffer (plb), by Promega (14 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (14 mentions) Pgl3 control vector, by Promega (13 mentions) Dual luciferase assay kit, by Promega (12 mentions) Pgl3 vector, by Promega (10 mentions) Pmirglo, by Promega (9 mentions) Dual luciferase assay, by Promega (9 mentions) Pgl3 promoter vector, by Promega (8 mentions) Luciferase assay system, by Promega (7 mentions) Pgl3 basic, by Promega (7 mentions) Dual luciferase reporter system, by Promega (6 mentions) Lipofectamine ltx, by Thermo Fisher Scientific (6 mentions)

Close spelling variants of this product

PRL‐TK‐Report vector PRL-TK Renilla luciferase control vector Renilla expression vector pRL‐TK PRL-TK renilla reporter vector PRL-TK Renilla controlluciferase reporter vector PRL-TK internal control vector PRL-TK Renilla luciferase internal control vector PRL-TK vector expressing Renilla luciferase PRL-TK reporter vector PRL-TK vector encoding Renilla luciferase

399 protocols using prl tk vector

Luciferase Assay for zbRIG-I and zbTRIM25

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HEK 293T cells, pre-seeded in 24-well plates, were transfected with 250 ng of pGL3-DrIFN 1-pro-Luc plasmid or pGL3-Basic empty vector with 25 ng of pRL-TK vector (Promega) together with pCMV-Myc-zbRIG-I or pCMV-Myc and pCMV-Flag or pCMV-Flag-zbTRIM25 (250 ng per well) for 24 h. Then, cells were incubated with poly I:C for 48 h and lysed. Luciferase activities were measured using the dual-luciferase reporter assay system (Promega). Relative luciferase activities were expressed as the ratio of firefly to Renilla luciferase activity. The results were the representative of three independent experiments in triplicate.
HEK 293T cells, pre-seeded in 24-well plates, were transfected with 250 ng of pGL3-DrIFN1-pro-Luc plasmid or pGL3-Basic empty vector with 25 ng of pRL-TK vector (Promega). Meanwhile, pCMV-Myc-zbTRIM25, mutant zbRIG-I or empty control plasmids were co-transfected. After being incubated with poly I:C for 48 h, cells were lysed for luciferase assay as described above. At least three independent experiments were performed.

Jin Y., Jia K., Zhang W., Xiang Y., Jia P., Liu W, & Yi M. (2019). Zebrafish TRIM25 Promotes Innate Immune Response to RGNNV Infection by Targeting 2CARD and RD Regions of RIG-I for K63-Linked Ubiquitination. Frontiers in Immunology, 10, 2805.

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Luciferase Assay for Transfected Stem Cells

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Transfections were done according to the Invitrogen protocol for Lipofectamine LTX & Plus reagent. For TSCs, a confluent 10 cm plate was split 1∶4 into a 24 well plate. We used a 1 µg∶4 µl ratio of DNA to reagent, and transfected 1 µg of reporter construct and 20 ng of pRL-TK vector (used as a transfection efficiency control vector, Promega Corp.) per well. Cells were lysed 24 hours post-transfection and frozen until luciferase assays were performed. For TGCs, plates were transfected 12 days after starting differentiation. We used a 1 µg∶3 µl ratio of DNA to reagent, and transfected 750 ng reporter construct and 15 ng of pRL-TK vector (used as a control vector, Promega Corp.). Cells were lysed 48 hours post-transfection and frozen until luciferase assays were performed. Each candidate was tested in triplicate within a single plate (technical replicates), and on at least 3 different days (biological replicates).

Tuteja G., Moreira K.B., Chung T., Chen J., Wenger A.M, & Bejerano G. (2014). Automated Discovery of Tissue-Targeting Enhancers and Transcription Factors from Binding Motif and Gene Function Data. PLoS Computational Biology, 10(1), e1003449.

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METTL14 and NLRP3 Regulatory Mechanisms

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METTL14 3′-UTR region carrying a putative miR-26a-5p binding site was inserted into the pGl3 vector. For METTL14 luciferase reporter assay, the HNPC were transfected with miR-26a-5p mimic and pGL3-METTL14-wild type (WT) or pGL3-METTL14-Mut plasmid (Mut) and pRL-TK vector (Promega, Madison, WI, USA) expressing the Renilla luciferase using Lipofectamine 2000. Otherwise, NLRP3 3′-UTR or 5′-UTR sequence was cloned into the pGl3 vector (Promega). HNPC transduced with METTL14 shRNA or overexpression vector were co-transfected with pGl3-NLRP3 3′-UTR or 5′-UTR luciferase reporter plasmid and pRL-TK vector (Promega) using Lipofectamine 2000 (Invitrogen). The relative luciferase activity was normalized to Renilla luciferase activity 48 h after transfection.

Yuan X., Li T., Shi L., Miao J., Guo Y, & Chen Y. (2021). Human umbilical cord mesenchymal stem cells deliver exogenous miR-26a-5p via exosomes to inhibit nucleus pulposus cell pyroptosis through METTL14/NLRP3. Molecular Medicine, 27, 91.

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Characterization of WFS1 Mutants

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Wild type (WT), p. W690fsX706 and p.F883X WFS1 cDNA were respectively synthesized and cloned into the pcDNA3.1 (+) vector by Generay Biotech (Shanghai, China). Sanger sequencing (ACGT, Wheeling, IL) was used to validate the sequence of the expression plasmids. HEK-293 T cells were seeded in 12-well plates, then transfected with 0.5 μg of ERSE-luciferase plasmid together with 1.0 μg of each WT, p.W690fsX706 or p.F883X WFS1 expression plasmid and 10 ng of pRL-TK vector (Promega, USA). After 24-h transfection, the cells were treated with or without thapsigargin (10 nmol/L) for 6 h. The assay was performed using the dual-luciferase reporter assay system (Promega, USA) according to the manufacturer’s instructions, and luciferase activities were normalized to Renilla luciferase values of the co-transfected pRL-TK vector.

Gong Y., Xiong L., Li X., Su L, & Xiao H. (2021). A novel mutation of WFS1 gene leading to increase ER stress and cell apoptosis is associated an autosomal dominant form of Wolfram syndrome type 1. BMC Endocrine Disorders, 21, 76.

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Genistein modulates Wnt signaling

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TOPflash luciferase assays were performed to assess the effect of genistein on the Wnt signalling pathway. After DMSO (4 days) or genistein (25 μM-4 days) treatment, cells were trypsinized and re-suspended in 48 well plates overnight. Cells were then transiently co-transfected with TOPflash (Upstate, Lake Placid, NY, USA) and pRL-TK Vector (Promega, Madison, WI, USA) encoding renilla luciferase as an internal control for transfection efficiency using lipofectamine LTX (Invitrogen). To look at the effect of miR-1260b on the beta-catenin dependent pathway, we knocked down miR-1260b with miR-1260b inhibitor (48 h transfection). Cells were trypsinized and re-suspended in 48 well plates overnight and then were transiently co-transfected with TOPflash (Upstate) and pRL-TK Vector (Promega).

Hirata H., Hinoda Y., Shahryari V., Deng G., Tanaka Y., Tabatabai Z.L, & Dahiya R. (2014). Genistein downregulates onco-miR-1260b and upregulates sFRP1 and Smad4 via demethylation and histone modification in prostate cancer cells. British Journal of Cancer, 110(6), 1645-1654.

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Luciferase Reporter Assay Protocol

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The cells were transfected with the indicated luciferase reporter in combination with the pRL-TK vector (Promega, Fitchburg, WI, USA) as an internal control, and the luciferase activities were determined using a microplate luminometer, as described in previous studies[30 (link)].

Hua X., Xu J., Deng X., Xu J., Li J., Zhu D.Q., Zhu J., Jin H., Tian Z., Huang H., Zhao Q.S, & Huang C. (2018). New Compound ChlA-F Induces Autophagy-dependent Anti-cancer Effect via Upregulating Sestrin-2 in Human Bladder Cancer. Cancer letters, 436, 38-51.

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Luciferase Reporter Assays for Transcriptional Regulation

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For luciferase reporter gene assays, cells were transfected with luciferase reporter plasmids (TOPFlash, FOPFlash, HOPFlash, HIPFlash, FHRE-Luc) 24 h prior to addition of indicated substances. An additional Renilla normalization plasmid was included during each transfection (pRL-TK vector, Promega). In case of additional siRNA transfection, the siRNA transfection was done an additional 24 h before luciferase reporter transfection. Treatment with indicated substances was 24h after which cells were lysed and firefly and Renilla luciferase activity was assayed in a multi-label plate reader (Envision 2102, Perkin Elmer) using the Dual Reporter Luciferase Assay System (E1960, Promega) according to the manufacturer’s recommendations.

Ma N., Wibowo Y.C., Wirtz P., Baltus D., Wieland T, & Jansen S. (2023). Tankyrase inhibition interferes with junction remodeling, induces leakiness, and disturbs YAP1/TAZ signaling in the endothelium. Naunyn-Schmiedeberg's Archives of Pharmacology, 397(3), 1763-1789.

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STAT5 Transcription Factor Assay

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TransAM^® STAT5 transcription factor assay kit was purchased from Active Motif (Carlsbad, CA). TurboFect Transfection Reagent was purchased from Thermo Scientific (Hudson, NH, USA). Dual-luciferase reporter assay system, pGL-STAT5 and pRL-TK vector were obtained from Promega Corporation (Promega, Madison, WI, USA). The plasmid encoding with STAT5B gene (pCMV6-AC-GFP-STAT5B) and anti-GFP antibody were purchased from OriGene Technologies (Rockville, MD, USA). Prolactin, ANTI-FLAG^® M2 Magnetic Beads, Monoclonal ANTI-FLAG^® M2 antibody and MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Liu L.J., Wang W., Kang T.S., Liang J.X., Liu C., Kwong D.W., Wong V.K., Ma D.L, & Leung C.H. (2016). Antagonizing STAT5B dimerization with an osmium complex. Scientific Reports, 6, 36044.

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Luciferase Assay in 293T Cells

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Luciferase reporter assay was performed in 293T cells using the reporter vector, pGL3-Promoter vector (cat#: E1761, Promega). The 35 bp S1606 and S961 fragments shown in Supplementary Table S1 were cloned into Sac I and Xho I sites in pGL3-Promoter vector individually (Supplementary Figure S1). For control, an irrelevant 35 bp fragment was cloned into the same vector. Constructs were transfected into 293T cells by FuGENE HD transfection reagent (Promega) together with an equal amount of the pRL-TK vector that provides constitutive expression of Relilla luciferase (Promega). The firefly luciferase reporter activity was normalizing to the Relilla luciferase activity using the Dual-Glo^® Luciferase Reporter Assay System (Promega). All experiments were performed according to the manufacturer's protocol. The data represent six independent biological replicates (n = 6).

Wu T., Jiang D., Zou M., Sun W., Wu D., Cui J., Huntress I., Peng X, & Li G. (2021). Coupling high-throughput mapping with proteomics analysis delineates cis-regulatory elements at high resolution. Nucleic Acids Research, 50(1), e5.

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Reverse Transfection of Luciferase Reporters

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RPTECs were seeded at a density of 1.5 × 10⁵/well in 24-well culture plates. Lipofectamine LTX regent (Invitrogen; Life Technologies) was used for reverse transfection of cells with 500 ng of the pGL4.12 reporter construct or with 200 ng of the pGL4.13 reporter constructs. A total of 10 ng of the pRL-TK vector (Promega) was also transfected as an internal control reporter. Cells were harvested 24 h after transfection and lysates were analyzed using the Dual-Luciferase reporter assay system (Promega). The ratio of firefly (expressed from pGL4.12 reporter constructs) to Renilla (expressed from pRL-TK) luciferase activity in each sample served as a measure of normalized luciferase activity.

Omata Y., Yamauchi T., Tsuruta A., Matsunaga N., Koyanagi S, & Ohdo S. (2021). RNA editing enzyme ADAR1 governs the circadian expression of P-glycoprotein in human renal cells by regulating alternative splicing of the ABCB1 gene. The Journal of Biological Chemistry, 296, 100601.

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