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Fig. 2. TGF-ß-induced nuclear translocation of GFPSmads is dependent upon continuous receptor signalling. (A) HaCaT cell lines stably expressing GFPSmad2, GFPSmad3 or GFPSmad4 were pretreated with cycloheximide and were then incubated with TGF-ß1 for 1 hour, followed by SB-431542 (7.5 µM) for up to 160 minutes. In the case of the GFPSmad4 cells, leptomycin B (LMB) was added 90 minutes after SB-431542 addition (150-minute time-point). Fluorescence images are shown at different time-points after initial TGF-ß treatment. Arrows indicate representative examples of cells demonstrating nucleocytoplasmic shuttling. For GFPSmad3 cells, the boxed region is shown magnified below to demonstrate that GFPSmad3 is partially excluded from the nucleoli upon TGF-ß treatment. Below are graphs showing quantitation of nuclear fluorescence, with fluorescence images collected every 3 minutes. The left-hand graph shows the average of the TGF-ß-induced nuclear fluorescence of the GFPSmad2 and GFPSmad4 cells marked with an arrow. Means and standard deviations are shown. The right-hand graphs show quantifications of the nuclear fluorescence for GFPSmad2 and GFPSmad4 throughout the whole experiment for one of the indicated cells in each case. (B) HeLa TK- cells were transiently transfected with plasmids expressing GFPSmad2 or GFPSmad4 together with FLAG-Smad2 and treated with cycloheximide, TGF-ß1, SB-431542 and LMB as in A. Fluorescence images are shown at different time-points after initial TGF-ß treatment. Arrows indicate representative examples of cells demonstrating nucleocytoplasmic shuttling. The punctate fluorescence observed in the cytoplasm of transiently transfected HeLa TK- cells is not seen in the stable HaCaT cell lines and thus seems to be a consequence of transient transfection. The experiments shown are representatives from at least three independent experiments.
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