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Interaction of NF-kB and IkBa, IkBaM, IkBa243N or IkBa244C studied with fluorescent fusion proteins by FRET in living cells
Author(s): Xian Li; Xiaojia Chen; Yonghong Tang
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Paper Abstract

In this paper, the location and interaction of NF-κB and IκBα (IκBαM, IκBα243N, or IκBα244C) in vivo is investigated by fluorescence resonance energy transfer (FRET). Co-transfection of a YFP-p65 construct with CFP- IκBα, C.FP-lid3aM (S32,36A), or CFP-IκBα243N(i-243) resulted in cytosolic localization of both proteins in almost all of the transfected cells. Co-transfection of YFP-p65 construct with CFP-Iw.Ba244C showed a predominant nuclear fluorescence of the proteins. The interaction between YFP-p65 and CFP-IκBα, CFP-IκBαM, CFP-IκBα243N or CFP-IκBα244C were further studied by acceptor bleaching experiments. When YFP-p65 were bleached, the fluorescence of CFP-IκBα, CFP-IκBm, CFP-IκBα243N increased. However, YFP-p65 and CFP-IκBα244C didn't have FRET and the fluorescence of CFP-IκBα244C were not influenced when YFP-p65 were bleached. This observation suggests that NF-κB interacted with the ankyrin repeat domain of IκBα, and our study domonstrates that the application of fluorescent fusion protein, FRET and acceptor bleaching technique to investigate protein-protein interactions in living cells might expand our understanding of these interactions considerably.

Paper Details

Date Published: 27 October 2006
PDF: 7 pages
Proc. SPIE 6047, Fourth International Conference on Photonics and Imaging in Biology and Medicine, 604739 (27 October 2006); doi: 10.1117/12.710928
Show Author Affiliations
Xian Li, South China Normal Univ. (China)
Xiaojia Chen, Jinan Univ. (China)
Yonghong Tang, South China Normal Univ. (China)

Published in SPIE Proceedings Vol. 6047:
Fourth International Conference on Photonics and Imaging in Biology and Medicine
Kexin Xu; Qingming Luo; Da Xing; Alexander V. Priezzhev; Valery V. Tuchin, Editor(s)

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