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Fig. 5. Mutation of NES2 reduces the export rate of Cdc25B3. Fluorescence loss in photobleaching (FLIP) of YFP-Cdc25B3, YFP-Cdc25B3(L62A), and YFP-Cdc25B3 together with leptomycin B (LMB) in G1 HeLa cells. Mitotic nocodazole-arrested HeLa cells were microinjected with expression vectors and released from nocodazole block. Six hours later, differential interference contrast (DIC) and YFP fluorescence images were acquired and cells were bleached in the cytoplasm for 45 seconds. The imaging and bleaching cycle was repeated 15 times. (A) Example of a FLIP experiment with YFP-Cdc25B3. Top four panels: Image before and after bleaching the right of the two daughter cells from the microinjected mitotic cell. The bleach area is circled in red. Bottom four panels: The same cells as above, after fixation and immunofluorescence with anti-GFP antibodies (red). (B) Quantification of FLIP. The nuclear fluorescence of the two daughter cells was quantified. After background subtraction, the value of nuclear fluorescence at the start of the experiment was normalised to 1. Measurements of nuclear fluorescence of the bleached cell relative to the unbleached cell are plotted in the graph. Each curve in the graph shows the average of six cells from three independent experiments (bars indicate standard deviation).
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