Fig. 7. Characterization of CRM1-independent shuttling of the STAT2 NES mutants STAT2-LL737/741AA. (A) C243 cells were transfected with STAT2-LL737/741AA-EYFP and subjected to cytoplasmic FLIP analysis. The bleach area is indicated with a white square in the first post-bleach image. The representative image series shows the fluorescence intensity in false color code (intensity increases from blue to red) before (0 sec) and after the consecutive bleaching periods. After the last bleach pulse subcellular distribution of STAT2-LL737/741AA-EYFP was monitored after another 300 seconds (+300 sec). (B) The nuclear fluorescence intensities of the bleached cells from A were measured in different experiments, normalized to the total fluorescence of the unbleached cells and plotted over time. Relative fluorescence intensities of STAT2-LL737/741AA-EYFP in the nucleus are shown. Colors of graphs reflect individual experiments. (C) C243 cells were transfected with either STAT2-LL737/741AA-YNES or STAT2-LL737/741AA-GNLS as indicated. Localization was determined in untreated cells by confocal analysis. The effect of LMB was monitored on STAT2-LL737/741AA-YNES localization 1 hour after treatment. (D) C243 cells expressing STAT2-LL737/741AA-YNES were treated for 4 hours with LMB (10 ng/ml) and subsequently subjected to FLIP analysis as described in A. Relative nuclear fluorescence intensities of STAT2-LL737/741AA-YNES from different experiments are shown. (E) C243 cells expressing STAT2-LL737/741AA-GNLS (left) or STAT1-GNLS (right) were pre-treated with cytochalasin D to induce bi-nuclear cells. The indicated area in one nucleus of a single cell was bleached until complete loss of fluorescence. Subsequently, recovery of unbleached STAT2-LL737/741AA-GNLS and STAT1-GNLS molecules into the nucleus was monitored for 10 minutes. Representative image-series show fluorescence intensities in false color code (increasing from blue to red) for the bleached cells (left) and neighboring cells (right) prior to bleaching and after the indicated post-bleach time periods.