This Small Business Innovation Research Phase I project focuses o the development of a two-Transcription factor NF-kB is activated by treatment with a diversity of stimuli, such as TNFa and IL-1b. Its activation leads to expression of an array of biologically important genes whose products contribute to cell growth, apoptosis, inflammation and immune response. Two methods are commonly used to monitor NF-kB activation; one is gel shift assay and the other is transcription induction of a reporter gene. Both of assays ar not suitable for high throughput drug screening because they are either time consuming or tedious. The proposal of this study is to develop a cell-based HT screening assay to detect NF-kB activation by monitoring IkB (degradation via its fusion with enhanced green fluorescent protein (EGFP). Our previous study indicates that EGFP can be converted from a stable protein into destablized by fusing with a degradation domain and can be used as a reporter for monitoring protein degradation. In this study, we propose to use EGFP to monitor IkB (degradation by making a fusion protein of these two proteins. Change of the fluorescence intensity of EGFP is expected to coordinate to the degradation of IkB( protein. Because IkB (degradation occurs within minutes after the treatment and EGFP detection is simple, this method will provide a quick and easy assay that allows to detect NF- kB activation. This technology will facilitate the discovery of potential drugs that can either stimulate or inhibit the process of NF-kB activation.
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| Li, X; Fang, Y; Zhao, X et al. (1999) Characterization of NFkappaB activation by detection of green fluorescent protein-tagged IkappaB degradation in living cells. J Biol Chem 274:21244-50 |
