Fig. 2. In vivo localization and interaction of EGR-3 or EGR-4 and NF-B p65 proteins. Jurkat T cells were transiently transfected with EGR-3-ECFP and p65-EYFP or EGR-4-ECFP and p65-EYFP expression vectors, and fluorescence images were recorded 24 hours after transfection. Images were acquired under CFP, YFP and FRET filter settings (top) or upon photobleaching of the acceptor protein (Bleach, bottom). EGR-3-CFP and EGR-4-CFP are localized predominantly to the nucleus and p65-YFP is present in the cytoplasm. Upon photobleaching of the acceptor, the fluorescence signal of the donor increases because cross-talk between the proteins is eliminated. The fluorescence signal of the acceptor and the FRET signals are completely abrogated. Staining of DNA with DAPI indicates the position of the nuclei and the Jurkat T cells are shown with digital interference contrast (DIC) microscopy. Bar, 10 µm.