Fig. 3. Controls for FLIP analysis. (A) HeLa TK^- cells were transiently transfected with plasmids expressing GFP, GFPRanBP1NES or GFPPKC. Cells were unstimulated except for those expressing GFPPKC which were stimulated with TPA. Cells were photobleached in the cytoplasm as indicated by the red diamond. The fluorescence images shown prior to photobleaching (time=0 seconds) and then after three consecutive 80-second bleaching periods. In all cases, the plane of focus was 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 right-hand graphs show the FLIP analysis. The fluorescence was quantitated at the bleach point and at a reporting point indicated by the blue square. The intensity of fluorescence is represented in arbitrary units. (B) Stimulation of cells with TPA induces the translocation of GFPPKC to the membrane. HeLa TK^- cells expressing GFPPKC were either untreated or treated with TPA for up to 10 minutes. Fluorescence images are shown. Here, the plane of focus is again a cross-section through the cell. The experiments shown are representatives from at least three independent experiments.