Fig. 4. The C-terminal nucleic-acid-binding region affects the mobility of the nucleoplasmin family proteins. (A) Diagram of nucleoplasmin family proteins. B23.2 and NPM3 lack the C-terminal nucleic-acid-binding region of B23.1. The position of the CK2 and CDK phosphorylation sites (Thr199, 219, 234 and 237) are indicated by an arrowhead and arrows, respectively. (B) Representative image showing distribution of GFP-B23.1, GFP-B23.2 and GFP-NPM3 in the nuclei of HeLa cells. The cells were transiently transfected with the corresponding plasmid and observed after 36 hours under a Leica confocal microscope. Relative amounts of B23.1, B23.2 and NPM3 present in the nucleolus and nucleoplasm are shown in the box. (C) Fluorescence recovery after photobleaching (FRAP) analysis of GFP-tagged nucleoplasmin family proteins. Cells were transfected with GFP-expressing construct and subjected to photobleaching 24-36 hours post transfection using a 488 nm laser. Five scans were taken before bleaching. An area in the nucleolus or a whole nucleolus was bleached and the fluorescence recovery of the bleached spot was measured every 5 seconds. Normalized fluorescence recovery in the ROI at each time point after photobleaching was plotted against time. The t_1/2s of recovery for the nucleoplasmin family proteins are indicated in the figure. Error indicates s.d. of the mean for each fitted curve.