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Fig. 2. FRAP and FLIP analyses reveal that the mobility of SR45 and SR1/SRp34 depends upon ATP. (A-D,F-I,K-L) A speckle (arrowhead) or a defined nucleoplasmic area (circle) in control or ATP-depleted cells was imaged before bleaching and during recovery for the time indicated. (A-D,F-I) Subsets of confocal images of a speckle (arrows in F) in ATP-depleted cells (A) SR45 and (F) SR1/SRp34, and control cells (B) SR45 and (G) SR1/SRp34. The entire set of time-lapse images of SR45 is animated in Movie 2, supplementary material. Subsets of confocal images of a nucleoplasmic area (circle) in ATP-depleted cells (C) SR45 and (H) SR1/SRp34, and control cells (D) SR45 and (I) SR1/SRp34. The entire time-lapse series of images for SR45 is animated in Movie 3, supplementary material. Bars, 5 µm. (E,J) Quantification of recovery kinetics of a speckle or a nucleoplasmic area in ATP-depleted cells of (E) SR45 and (J) SR1/SRp34. Curves shown are averages of at least ten nuclei in three different experiments. Error bars are ± s.e.m. (K,L) Subset of images of FLIP analyses of SR45. In each panel two adjacent nuclei of GFP-SR45 cells, either (K) untreated or (L) treated with NaN₃ and 2-deoxyglucose are shown. A defined area (circle) in the lower nucleus of each panel was repeatedly bleached and photographed at the indicated times. The upper nucleus was not bleached and served as a reference for the normalization of fluorescence intensities. Very little loss of fluorescence in the unbleached zone of ATP-depleted nuclei indicates that the movement of GFP-SR45 is dependent upon ATP. The entire dataset of K and L is animated in Movie 4, supplementary material. (M) Quantitative FLIP analyses of the control and ATP-depleted GFP-SR45. Data presented are representative of five nuclei.

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