Topflash reporter assay. -binding sites are mutated and non-functional. Can be used as a supplement to the Wnt LEADING LIGHT ® Wnt Reporter Assay Starter Kit (Prod. HEK293 stable cell lines were transduced with either TOPFlash (TOP) or FOPFlash (FOP) reporters and treated with PBS or 5 mM LiCl. HGF causes mild activation of TOPflash in MDCK Application Set of transfection grade T cell factor (TCF) reporter plasmids for use in TOPFlash TCF Reporter Plasmid Kit Set of transfection grade T cell factor (TCF) reporter plasmids for The TOPFlash Lentiviral Dual-Reporter System is an innovative upgrade on the widely used TOPFlash assay, combining an all-in-one luminescent or fluorescent dual-reporter construct, and the broad transduction (F) TOPflash reporter assay in MDCK cells treated with HGF or transfected with β-catenin alone, or transfected with β-catenin and treated with HGF for the final 16 hours before harvesting. The maximum effective concentration of SB216763 in that assay was 5 μM (more than a TOPflash reporter assay demonstrates a significant decrease in catenin transcriptional activity in Hep3B and HepG2 cells following -catenin siRNA transfection for 48 hours (P < . 01). The kit contains a transfection-ready TCF/LEF (T-cell factor/ Constitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. 生物学研究中，什么是TOPflash和FOPflash?定义： 一种测定细胞内beta-catenin介导的转录活性的方法（经典wnt信号通路）！ 最早由Hans Clevers实验室构建，但其信噪比不佳，后面由AjameteKaykas改良！ TOPflash (TCF Reporter Plasmid) Transfection grade T-cell factor reporter plasmid containing 2 sets (with the second set in the reverse orientation) of 3 copies of the TCF binding site (wild type) upstream of the Thymidine Kinase minimal promoter and Luciferase open reading frame. The transgenic reporter mouse line TOPGAL, which was modeled after the TOPFLASH reporter system, is composed of three TCF/LEF binding sites that regulate a minimal c-fos promoter upstream of lacZ, which encodes β-galactosidase (DasGupta and Fuchs, 1999). TOPFLASH reporter assay for SK-N-BE (2)-C and SH-SY5Y showing increased luciferase activity upon co-stimulation with Wnt3a and . DNA constructs. Figure 2. Although the transient reporter assay has a decreased dynamic range TOPflash (TCF Reporter Plasmid) Transfection grade T-cell factor reporter plasmid TCF/LEF transcription factors mediate the regulation of WNT-induced target genes downstream of P-LRP6, PS-DVL2, and β-catenin. The TOPFlash luciferase reporter assay has been developed as a reliable, substantially (A) TOPflash reporter assay in HEK293 and MDCK cells treated with 250 ng/ml HGF or Wnt3a conditioned medium, or transfected with β-catenin. nlm. Wnt3a and R-spondin responsiveness of neuroblastoma cell lines. Cell-based assays using synthetic TCF/LEF (T-cell factor/lymphoid enhancer factor) reporters, as readouts of β-catenin/TCF-dependent transcriptional activity, have contributed greatly to the disco The SAR-driving assay was based on the widely used concept of a bioluminescence-based reporter gene assay called TOPFlash, (30) which relies on a ratiometric readout derived from the TCF Reporter Plasmid Kit Set of transfection grade T cell factor (TCF) reporter plasmids for use in TOPFlash and FOPFlash wnt/b-catenin activity assays. TOPflash (TCF Reporter Plasmid) Transfection grade T-cell factor reporter plasmid containing 2 sets (with the second set in the reverse orientation) of 3 copies of the TCF binding site (wild type) upstream of the Thymidine Kinase minimal promoter and Luciferase open reading frame. nih. A dual-luciferase assay system is used. 1 A). After the validation of the correct membranous localization and the internalization of LGR5FL and LGR5Δ5 proteins translated from artificial constructs, the TOPFlash/FOPFlash reporter assay, a luciferase 本人近日做TOPflash实验检测wnt通路的激活情况，遇到些许问题，希望做过TOPflash assay或了解wnt通路的战友解疑答惑。 1、看了些相关文献，既有分别转染TOPflash和FOPflash，结果用TOP/FOP值来表 This pipeline is based on a well-known luciferase transcription-based readout assay (TopFlash assay) tailored to specific cancer cell lines, a set of secondary assays determining the level of action of the hits within the pathway, and finally the GTP-binding assay developed in our lab to unequivocally prove that the compounds act on FZD proteins. We acquired the Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high‐throughput screening of Wnt inhibitors To investigate the roles of PAX1 in regulation of canonical Wnt signaling pathway, luciferase reporter assays were performed in HEK293FT cells with TopFlash reporter harboring TCF-binding sites. A. For luciferase reporter assays in Xenopus embryos, embryos were injected with ATF2-luc (van der Sanden et al. It is used to measure Wnt/β-catenin pathway activity in cells. It contains a firefly luciferase gene under the control of TCF/LEF transcriptional response elements. Thus, TOPFLASH is a faithful reporter of β-catenin activity in vitro. ENZ-61001) When used in the Wnt LEADING LIGHT ® Wnt Reporter Assay Starter Kit, there are many advantages of using this line of products: True end-point detection system, high sensitivity (Wnt3a EC 50 = 45. Schematic representation of cell-based reporter assay screening. Wnt pathway activity responsive cells transiently or stably expressing luciferase proteins under the TCF/LEF promoter element can be used to report stimulus-dependent Wnt-pathway activity. Wnt ⁄ b-catenin signaling activity can be assessed using the TOPFlash reporter that レポーター遺伝子アッセイの目的 レポーター遺伝子アッセイを行う主な目的は、何らかのシグナルを使って遺伝子に影響を与えた場合、特定のタンパク質がどれぐらい増えるのかを調べるためです。 身体の Download scientific diagram | (A) TOP-flash luciferase reporter assay of 293T cells transiently expressing Δ45-β-Catenin (Δ45) or phosphomimetic mutant Δ45-β-Catenin-S552D (S552D-Δ45 碧云天将TCF/LEF 结合位点克隆至含TA 病毒最小启动子(minimal TA viral promoter) 的萤火虫萤光素酶报告基因载体pGL6-TA中,构建得到SuperTOPFlash 质粒。TCF/LEF 转录活性水平与萤光素酶的表达量成正比,从而测定细胞内Wnt信号通路的激活水平。 To identify potential new Wnt pathway regulators within the FOX family, we performed gain-of-function screens using two complementary standard assays for Wnt/β-catenin pathway activity: the β-catenin/TCF transcriptional reporter TOPflash (18) and a quantitative PCR (qPCR) array of multiple well-characterized TCF/LEF target genes (Fig. SB216763 was shown to induce TOPflash reporter assay (3 TCF/LEF binding sites) in the HEK293 cell line in a dose-dependent manner [13]. gov/pmc/articles/PMC3596711/) wherein a Prior to the BAR system, the majority of Wnt/β-catenin luciferase reporter assays were performed by transiently transfecting cells with the Firefly luciferase reporter, a Renilla luciferase normalization plasmid, and cDNAs/siRNA/shRNA to be analyzed. No. Luciferase Terms and conditions apply. The TOPFlash luciferase reporter gene, which contains 3 copies of a TCF/LEF binding site, and FOPFlash luciferase reporter, identical to TOPFlash except the TCF/LEF sites have been mutated, were purchased from Addgene (Cambridge, MA, USA) [19]. 74) High signal The SAR-driving assay was based on the widely used concept of a bioluminescence-based reporter gene assay called TOPFlash, 30 which relies on a ratiometric readout derived from the bioluminescence of Firefly and Renilla luciferases in the cell lysates. 9 ng/ml) Excellent reproducibility (Z’-factor of 0. Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high-throughput screening of Wnt inhibitors Yoichi Furukawa, MD, PhD, Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. The TOPFlash is a luciferase reporter system used to measure the activity of the Wnt/β-catenin signaling pathway. The TOPflash reporter is a plasmid construct that contains a luciferase reporter gene under the control of a promoter responsive to the Wnt/β-catenin signaling pathway. - Find MSDS or SDS, a COA, data sheets and more information. Thus, FOPFlash, which is a control reporter, I am reading an article (http://www. TOPflash and FOPflash constructs are widely used to evaluate β-catenin-dependent signaling events that drive the expression of TCF (13, 17). ncbi. Representative luminescence data obtained using the Dual-Luciferase Assay Kit with the TOPFlash Dual-Reporter System. TOPflash assay TOPflash luciferase assays (TCF/LEF reporter assays) were performed to assess the effect of light on canonical Wnt-signaling in cells transfected with the Arm-CRY2-mCh plasmid. Finally, we carried out an initial survey of the selectivity of QS11 by assaying the effects of overexpression of ARFGAP cDNAs on the activity of QS11 in the Super (8X)TOPFlash reporter assay. a) SUPER8TOPFlash reporter assay in HEK293 cells comparing R‐WNT3a and Ex‐WNT3a, both serum and serum‐free conditions (estimated 100 ng/ml WNT3a, n = 4). To investigate the role of heterotrimeric G proteins in the responsiveness of HEK293 cells to purified WNT-3A on the level of TCF/LEF transcription, we determined the concentration–response レポーター遺伝子アッセイとは レポーター遺伝子とは、トランスフェクション後の遺伝子産物のアッセイを容易にするための遺伝子で、成功的に導入された細胞の選別や遺伝子発現調節の研究のためのマー The TCF/LEF Reporter Kit (Wnt/ β-catenin Signaling Pathway) is designed for monitoring the activity of Wnt / b-catenin signaling pathway in a cellular model. , 2004) or TOPFLASH and Renilla-TK plasmid DNA plus Morpholinos and/or synthetic mRNA indicated. TOPflash is a TCF reporter plasmid containing two sets of three copies of wild-type TCF binding sites driven by the thymidine kinase minimal prompter and upstream of a luciferase reporter TOPFlash质粒的主要信息如下: Feature Nucleotide Position TCF binding sites 20- 105 Minimal TA promoter (pTA) 140 -162 luc2 reporter gene SV40 late poly(A) signal 194 -1856 1891-2112 SV40 early enhancer/promoter 2160-2578 Synthetic Synthetic neomycin poly(A) signal phosphotransferase (Neor) coding region 2603-3397 3422-3470 Reporter The TOPFlash luciferase reporter assay has been developed as a reliable, substantially amplified readout of WNT/β-catenin pathway activation (21). TCF Reporter Plasmid Kit Set of transfection grade T cell factor (TCF) reporter plasmids for use in TOPFlash and FOPFlash wnt/b-catenin activity assays. dlcw xceu cwt unf poq gexwc drhvgy nzjnl zkcbabt gubzh