NFkappaB DD Red Reporter System
NFkappaB DD Red Reporter System
Brand: Takara Bio.
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NFkappaB DD Red Reporter System
The NFkB DD Red Reporter System is designed to monitor NFkB activation in mammalian systems, with minimal background signal. It includes the pNFkB-DD-tdTomato Reporter vector and Shield1.
pNFkB-DD-tdTomato encodes a red fluorescent protein reporter tagged at its N-terminus with the ProteoTuner destabilization domain (DD), and under the control of the NFkB promoter. The DD causes the DD-tdTomato reporter to be rapidly targeted to and degraded by proteasomes. This minimizes background fluorescence from leaky promoters prior to promoter activation.
To monitor NFkB activity, a candidate inducer is added to the medium simultaneously with the DD's stabilizing ligand, Shield1. This allows DD-tdTomato to accumulate in response to NFkB activation. As a result, only the reporter molecules expressed during NFkB induction contribute to the fluorescence signal. This system provides a considerably higher signal-to-noise ratio than can be obtained with non-destabilized or constitutively destabilized reporter systems.
DD-Fluorescent Protein promoter reporters provide a much greater fold increase in signal intensity than traditional fluorescent protein reporters, which do not contain the DD
DD-Fluorescent Protein promoter reporters provide a much greater fold increase in signal intensity than traditional fluorescent protein reporters, which do not contain the DD. HEK 293 cells were transfected with plasmids encoding the following reporters: CRE-tdTomato, CRE-DD-tdTomato, CRE-ZsGreen1, and CRE-DD-ZsGreen1. 24 hr later, the cells were stimulated with 10 μM forskolin and simultaneously treated with 1 μM Shield1. After 4.5 hr, fluorescence intensity was measured via flow cytometry, and fold induction was calculated. The tdTomato and ZsGreen1 reporters containing the DD had three- and six-fold greater fluorescence intensity respectively, than the versions without the DD, due to the latter’s increased background levels.
tdTomato, but not GFP, can be detected in the SCID mouse cadaver phantom model
tdTomato, but not GFP, can be detected in the SCID mouse cadaver phantom model. False-color overlay images (regions of interest encircled) indicate that the imaging system could detect tdTomato fluorescence in the cadaver model, but not GFP fluorescence. Panel A. Implanted tube with 100 x 106 MDA-MB-231-tdTomato-expressing cells, imaged with the DsRed filter set. Exposure time: 1 sec. Panel B. Implanted tube with 100 x 106 MDA-MB-231-GFP-expressing cells, imaged with the GFP filter set. Exposure time: 1 sec.
Ligand-dependent, targeted, and reversible fluorescent protein reporter stabilization
Ligand-dependent, targeted, and reversible fluorescent protein reporter stabilization. A small destabilization domain (DD) is fused to the fluorescent protein reporter. The small membrane-permeant ligand Shield1 binds to the DD and protects the entire fusion protein from proteasomal degradation. Removal of Shield1, however, causes rapid degradation of the entire DD-fluorescent protein reporter. The default pathway for the DD-Fluorescent Protein Reporter Systems is degradation of the DD-fluorescent protein reporter, assuring low background, unless Shield1 is present, assuring low background.
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DD-Fluorescent Protein promoter reporters provide a much greater fold increase in signal intensity than traditional fluorescent protein reporters, which do not contain the DD
DD-Fluorescent Protein promoter reporters provide a much greater fold increase in signal intensity than traditional fluorescent protein reporters, which do not contain the DD. HEK 293 cells were transfected with plasmids encoding the following reporters: CRE-tdTomato, CRE-DD-tdTomato, CRE-ZsGreen1, and CRE-DD-ZsGreen1. 24 hr later, the cells were stimulated with 10 μM forskolin and simultaneously treated with 1 μM Shield1. After 4.5 hr, fluorescence intensity was measured via flow cytometry, and fold induction was calculated. The tdTomato and ZsGreen1 reporters containing the DD had three- and six-fold greater fluorescence intensity respectively, than the versions without the DD, due to the latter’s increased background levels.
tdTomato, but not GFP, can be detected in the SCID mouse cadaver phantom model
tdTomato, but not GFP, can be detected in the SCID mouse cadaver phantom model. False-color overlay images (regions of interest encircled) indicate that the imaging system could detect tdTomato fluorescence in the cadaver model, but not GFP fluorescence. Panel A. Implanted tube with 100 x 106 MDA-MB-231-tdTomato-expressing cells, imaged with the DsRed filter set. Exposure time: 1 sec. Panel B. Implanted tube with 100 x 106 MDA-MB-231-GFP-expressing cells, imaged with the GFP filter set. Exposure time: 1 sec.
Ligand-dependent, targeted, and reversible fluorescent protein reporter stabilization
Ligand-dependent, targeted, and reversible fluorescent protein reporter stabilization. A small destabilization domain (DD) is fused to the fluorescent protein reporter. The small membrane-permeant ligand Shield1 binds to the DD and protects the entire fusion protein from proteasomal degradation. Removal of Shield1, however, causes rapid degradation of the entire DD-fluorescent protein reporter. The default pathway for the DD-Fluorescent Protein Reporter Systems is degradation of the DD-fluorescent protein reporter, assuring low background, unless Shield1 is present, assuring low background.
