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Fig. 6. FLIP/FRAP analysis reveals a TGF-ß-dependent change in mobility of GFPSmad2 and GFPSmad4 in the nucleus. HeLa TK- cells were transiently transfected with plasmids expressing GFPNLS, GFPß-galactosidaseNLS, GFPPKC
, GFPSmad2 or GFPSmad4 with FLAG-Smad2. Cells were either unstimulated or pretreated for 1 hour with LMB or TGF-ß1 as indicated. The cells expressing GFPPKC were pretreated for 10 minutes with TPA. Prior to bleaching, eight measurements of fluorescence were taken at the bleach point in the nucleus (red square) over a period of 2 seconds, then cells were bleached for 11 seconds. The recovery of fluorescence in the bleached area and fluorescence loss at a distant point also in the nucleus (blue square) were measured at 250-millisecond intervals. The levels of fluorescence were normalised to the mean of fluorescence registered before the photobleaching. In all cases, the plane of focus is a cross-section through the cell, except for the TPA-induced cells expressing GFPPKC where the plane of focus was in the plasma membrane. The data are representatives from three different experiments.
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