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Fig. 7. Location and mobility of the CDK1 phosphorylation-mimicking mutant. (A) Location of T4E-GFP mutant in interphase HeLa cells. T4E-GFP transfected cells were analyzed after 36 hours under a fluorescence microscope. This mutant shows a similar distribution in the nucleus to the nucleoplasmin family proteins, B23.2 and NPM3 lacking the C-terminus. Bar, 10 µm. (B) Expression of T4E-GFP mutant in interphase HeLa and CMT3 cells. The western blot was developed with anti-GFP monoclonal antibody. (Lane 1) untransfected cell lysate; (lane 2) T4E-GFP transfected HeLa cell lysate; (lane 3) T4E-GFP transfected CMT3 cell lysate. (C) Fluorescence recovery after photo bleaching (FRAP) analysis of GFP-tagged Thr199/219/234/237 Glu mutant B23. 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 T4E-GFP mutant and wild-type protein B23-GFP are indicated in the figure. Error indicates s.d. of the mean for each fitted curve.

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