NFkappaB DD Cyan Reporter System
NFkappaB DD Cyan Reporter System
Brand: Takara Bio.
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NFkappaB DD Cyan Reporter System
The NFkB DD Cyan Reporter System is designed to monitor NFkB activation in mammalian cells, with minimal background signal. It includes the pNFkB-DD-AmCyan1 Reporter vector and Shield1.
pNFkB-DD-AmCyan1 encodes a cyan 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-AmCyan1 reporter to be rapidly targeted to and degraded by proteasomes. This minimizes background signal 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-AmCyan1 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.
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.
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.
