Fig. 7. Cell-cycle dependent immobilization of GFP-Ciz1. (A) Histogram shows the proportion of transfected cells that retain GFP-fluorescence in the nucleus after extraction with Triton X-100. Twenty-four hours after transfection, full-length ECiz1 is retained in all transfected cells, GFP-ECiz1 N471 in virtually none, whereas GFP-C275 and GFP-708-830 was retained in 27% and 29%, respectively. (B) The proportion of transfected cells with immobilized GFP-C275 increases to almost 80% by 48 hours after transfection, but this is significantly reduced when cells are prevented from cycling during the second 24 hours by exposure to thymidine. (C) The proportion of cells with immobilized GFP-C275 increases during late G1 or early S phase. Cells were arrested with thymidine for 24 hours, released for 10 hours then given a second thymidine block for a further 14 hours. Arrested cells were transfected with GFP-C275 then released for the indicated times. The cell cycle profile of each population is shown. (D) The proportion of cells with immobilized GFP-C275 increases during late G1 or early S phase, but remains constant in populations arrested for a further 10 or 22 hours in S phase. Transfection frequencies were the same at the beginning and end of the time course (not shown). Histograms show the average of three data sets with standard errors.