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Fig. 6. Quantitative FRAP analysis of full-length and mutant NRF-GFP. (A) C243 cells stably transfected with expression plasmids encoding the full-length NRF-GFP were subjected to quantitative FRAP analysis. The area of an entire nucleolus was bleached and fluorescence recovery kinetics for the bleached region and change of fluorescence intensity in an unbleached nucleolus were measured. To allow a quantitative comparison of signal loss in unbleached areas with signal gain in the bleached area, plotted data were not corrected for the overall loss of fluorescence induced by the measurements of fluorescence intensity. For clarity, only selected time points are marked by a symbol. Open symbols indicate the bleached area, whereas closed symbols mark the area of unbleached nucleoli. Each graph represents the average of data from ten single cells. (a) Fluorescence recovery kinetics for NRF-GFP were compared in C243 cells (squares) and in a stably transfected NIH3T3 cell line (triangles). (b) Fluorescence recovery kinetics for NRF-GFP at room temperature (squares) and at 37°C (triangles) were compared in C243 cells. (c) One part of a nucleolus was bleached and fluorescence recovery kinetics for the bleached region (open squares) and change of fluorescence intensity in the unbleached area (closed circle) of the same nucleolus were measured. (B) Kinetics of fluorescence recovery for NRF-GFP and NRF^362-690-GFP were measured both in the nucleoplasm (a,b) and in the cytoplasm (c,d). Bleaching was done in a circular area and fluorescence recovery for the bleached region and change of fluorescence intensity in an unbleached area of the same size were plotted over time. For clarity, the scale of the y-axis was changed. (a,c) NRF-GFP; (b,d) NRF^362-690-GFP. (C) C243 cells transfected with expression plasmids for the indicated GFP-tagged NRF deletion mutants were subjected to quantitative FRAP analysis. The overall FRAP settings are described in (A). (a) NRF^1-550-GFP, (b) NRF^1-480-GFP, (c) NRF^1-434-GFP, (d) NRF^1-402-GFP, (e) NRF^1-361-GFP, (f) FRAP analysis of NRF^1-361-GFP in the nucleoplasm (np), (g) NRF^362-690-GFP, (h) Comparison of the FRAP kinetics for full-length NRF and the indicated deletion mutants.

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