Abstract
A highly fluorescent mutant form of the green fluorescent protein (GFP) has been fused to the human nuclear factor kappaB (NF-kappaB) p50 and p105 (p50/IkappaB gamma), a precursor protein of NF-kappaB p50. GFP-p50 and GFP-p105 were expressed in monkey COS-7 cells and human HeLa cells. Translocation of these chimeric proteins was observed by confocal laser scanning microscopy. GFP-p50 (without IkappaB gamma) in the transfected cells resided in the nucleus. On the other hand, GFP-p105 (GFP-p50 with IkappaB gamma) localized only in the cytoplasm before stimulation and translocated to the nucleus with stimulant specificity similar to that of native NF-kappaB/IkappaB. In addition, the translocation of NF-kappaB to the nucleus had a distinct lag time (a quiescent time) in the target cells. The lag time lasted 10-20 min after stimulation with hydrogen peroxide or tumor necrosis factor alpha. It was suggested that this might be due to the existence of a limiting step where NF-kappaB is released from NF-kappaB/IkappaB by the proteasome.
MeSH terms
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Amino Acid Substitution
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Animals
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Blotting, Western
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COS Cells
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Cell Nucleus / drug effects
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Cell Nucleus / metabolism*
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Cytoplasm / drug effects
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Cytoplasm / metabolism
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DNA-Binding Proteins / metabolism
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Green Fluorescent Proteins
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HeLa Cells
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Humans
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Hydrogen Peroxide / pharmacology
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I-kappa B Proteins
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Luminescent Proteins / biosynthesis
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism*
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Microscopy, Confocal
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NF-kappa B / biosynthesis
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NF-kappa B / metabolism*
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NF-kappa B p50 Subunit
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Protein Precursors / biosynthesis
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Protein Precursors / metabolism
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Recombinant Fusion Proteins / biosynthesis
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Recombinant Fusion Proteins / metabolism
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Scyphozoa
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Time Factors
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Transcription Factors / biosynthesis
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Transcription Factors / metabolism
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Tumor Necrosis Factor-alpha / pharmacology
Substances
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DNA-Binding Proteins
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I-kappa B Proteins
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Luminescent Proteins
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NF-kappa B
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NF-kappa B p50 Subunit
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NFKB1 protein, human
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Protein Precursors
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Recombinant Fusion Proteins
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Transcription Factors
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Tumor Necrosis Factor-alpha
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Green Fluorescent Proteins
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Hydrogen Peroxide