Fig. 7. Nuclear translocation of IFN requires the presence of the C-terminus NLS. (A) WISH cells, transduced for 8 hours with an empty vector control (top) or a vector expressing non-secreted IFN (middle left) or NLS-modified IFN (middle right) were probed with a monoclonal antibody to IFN and stained with a Alexa-Fluor-488-conjugated secondary antibody and analyzed by confocal microscopy. The lower graphs show mean fluorescence intensity comparisons for non-secreted IFN and NLS-mutated IFN. Measurements of fluorescence intensities were done with IP Lab (Scanalytics) using the MP-line measure tool. Mean pixel intensity was measured across a line drawn through the cells in the plane of the images shown. A representative measurement line is shown in white. The resultant graphs were generated by the software averaging the intensities of a total of 100 pixels on either side of the line at each point along the line. Triplicate determinations showed a significance of P<0.25 (t-test) for nuclear presence of IFN versus NLS mutant. (B) Quantitation of images. Images of cells transduced with non-secreted IFN or NLS-mutated nonsecreted IFN were used to determine Fn/Fc values, which are shown in columns 1 and 2, respectively. Seven fields were examined and the results were averaged±s.d. The significance was P<0.02 by the t-test. Calculations for fluorescence in the nucleus (N) versus cytoplasm (C) were also done by using N/N+C. Four independent measurements showed a P<0.025 for the nuclear translocation of IFN for the wild-type IFN versus the NLS-mutated IFN.