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Fig. 4. Dynamics of nucleolar targeting of NRF-GFP. (A) C243 cells stably transfected with NRF-GFP were subjected to FRAP analysis. The area of an entire nucleolus was bleached (indicated by an arrow in the first post-bleach panel) and images collected before and at the indicated time points after the end of the bleach pulse are shown. (B) Quantitative data of fluorescence recovery kinetics for NRF-GFP were recorded and plotted over time. The fluorescence intensities in bleached and unbleached nucleoli were measured for two adjacent cells (shown below; the bleached nucleolus is indicated by an arrow in the pre-bleach panel). Plotted data were not corrected for the overall loss of fluorescence induced by the image collection, to allow a quantitative comparison of signal loss in unbleached areas with signal gain in the bleached area. The FRAP rate of the bleached nucleolus (number 1) is represented by red diamonds; 2 (green squares) and 3 (blue triangles) are unbleached nucleoli of the same cell; 4 (orange circles) and 5 (magenta squares) are nucleoli of an adjacent cell. (C) C243 cells expressing NRF^1-361-GFP were subjected to FRAP analysis. The area of an entire nucleolus was bleached (indicated by an arrow in the first post-bleach panel) and images collected before and at the indicated time points after the end of the bleach pulse are shown. (D) Quantitative data of fluorescence recovery kinetics for NRF^1-361-GFP were recorded and plotted over time. Fluorescence intensities of bleached and unbleached nucleoli as well as in nucleoplasmic areas were determined. The FRAP rate of the bleached nucleolus (1) is indicated by red diamonds; 2 (green squares) and 3 (blue triangles) are unbleached nucleoli of the same cell; 4 (orange circles) is the nucleoplasm.

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