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Fig. 3. Fluorescence loss in photobleaching (FLIP) proves that the putative first NES is not active. 293 cells were grown on coverslips and transfected (top) with YFP-NIK(1-794), which contains the first predicted NES, or with (bottom) YFP-NIK(1-769) lacking this domain. The coverslips were mounted onto an incubation chamber filled with medium and live cells were examined by laser scanning microscopy. The cytosol was bleached with high laser power and cytosolic as well as nuclear fluorescence intensities were recorded over time as described in the Materials and Methods section. Fluorescence decreased significantly in the cytosol whereas it remained constant in the nucleus for both constructs indicating that nuclear NIK did not shuttle into the cytosol.
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