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

摘要

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, CFP-IκBαM (S32,36A), or CFP-IκBα243N(1-243) resulted in cytosolic localization of both proteins in almost all of the transfected cells. Co-transfection of YFP-p65 construct with CFP-IκBα244C 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.