Fig. 1. Photobleaching analysis of the efflux of GFP:STATc from the nucleus. (A) Principle of the photobleaching method. Isolated cells transformed with GFP:STATc, a blasticidin-based, `single copy' vector (Fukuzawa et al., 2001) in which there was a low to moderate fluorescence signal in the nucleus, were photobleached to reduce only the cytoplasmic fluorescence (the nucleus was masked) and then incubated in either the presence or the absence of 100 nM DIF for 5 minutes. Many of the cells in clumps, and a sub-set (approx. 10%) of the spatially separated cells, show a very high intrinsic level of Dd-STATc nuclear fluorescence. There is a DIF-independent, stress-induced mechanism that directs nuclear accumulation of Dd-STATc (T. Araki, M. Tsujioka, T. A. and J. G. W., unpublished data) and this may account for these `high background' cells. In such cells, nuclear fluorescence is retained irrespective of the presence or absence of DIF and they were not therefore included in the analysis. (B,C) Analysis of nuclear efflux rates of (B) GFP:STATc and (C) GFP:STATa in the presence and absence of DIF. Cells transformed with GFP:STATc or GFP:STATa were photobleached as described in A. The fluorescence signal from the cytoplasm and nucleus was monitored over 5 minutes. In order to maintain constant conditions, the `contrast-stretch' function of the microscope was turned off. In each experiment a total of at least 30 cells was analysed and the graph shows the ratio (nuclear signal — background)/(cytoplasmic signal — background) x 100 (%) ± s.d.

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