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Fig. 4. FLIP analysis of GFPSmad2 and GFPSmad4 proves nucleocytoplasmic shuttling. (A) HaCaT cell lines stably expressing GFPSmad2 or GFPSmad4 remained either unstimulated (i,iii) or were treated for 1 hour with TGF-ß1 (ii,iv). The bleach region in the cytoplasm is indicated (red diamond). Each row shows the fluorescence image prior to bleaching (time=0 seconds) and after three consecutive 80-second bleaching periods. The fluorescence was quantitated at the bleach point, at a reporting point in the nucleus of the same cell (blue square) and at a reporting point in the nucleus of an adjacent cell (white triangle). In each case the fluorescence was normalised to the initial fluorescence prior to photobleaching, and the relative fluorescence was plotted. Note that in unstimulated cells expressing GFPSmad4, the cell to the right of the bleached cell also bleaches because the bleach point also contacts the cytoplasm of that cell. The data are representatives from at least three different experiments.
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