Interaction of NF-kB and IkBa, IkBaM, IkBa243N or IkBa244C studied with fluorescent fusion proteins by FRET in living cells

Xian Li; Xiaojia Chen; Yonghong Tang

doi:10.1117/12.710928

27 October 2006 Interaction of NF-κB and IκBα, IκBαM, IκBα243N or IκBα244C studied with fluorescent fusion proteins by FRET in living cells

Xian Li, Xiaojia Chen, Yonghong Tang

Author Affiliations +

Proceedings Volume 6047, Fourth International Conference on Photonics and Imaging in Biology and Medicine; 604739 (2006) https://doi.org/10.1117/12.710928
Event: Fourth International Conference on Photonics and Imaging in Biology and Medicine, 2005, Tianjin, China

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.

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Xian Li, Xiaojia Chen, and Yonghong Tang "Interaction of NF-κB and IκBα, IκBαM, IκBα243N or IκBα244C studied with fluorescent fusion proteins by FRET in living cells", Proc. SPIE 6047, Fourth International Conference on Photonics and Imaging in Biology and Medicine, 604739 (27 October 2006); https://doi.org/10.1117/12.710928

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KEYWORDS

Proteins

Fluorescence resonance energy transfer

Luminescence

Image filtering

Current controlled current source

Confocal microscopy

Molecules

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