Fig. 5. Mobility of the nucleolar c-Myc-GFP in proteasome inhibitor treated cells. (A) FLIP analysis demonstrates that c-Myc-GFP shuttles between the nucleolus and the nucleoplasm/cytoplasm after proteasome inhibition. A cell expressing c-Myc-GFP (outlined by white dashed line) was treated with MG132 for 3 hours before being repeatedly bleached in a defined area in the cytoplasm (white circle). Confocal images of the cell were taken before bleaching and then at 2 second intervals. The panel shows images taken at the indicated time points. Bar, 20 µm. (B) FRAP analysis demonstrates a relatively stable interaction of c-Myc-GFP with a nucleolus after proteasome inhibition. A cell expressing c-Myc-GFP was treated with ALLN for 4 hours before being bleached in part of the nucleolus for 1 second (white circle). Confocal images were taken before bleaching and then every 120 seconds. The panel shows images taken at the indicated time points. Bar, 20 µm. (C) Quantitative measurements of the fluorescence recovery. The curve indicates the mean value for the relative fluorescence intensity plotted as a function of time. The error bars represent the standard deviation (n=5 cells). The fluorescence was recovered within 30 minutes and the mobile fraction was calculated to 84%.

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