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Fig. 7. FRAP analyses reveal two pools of wt GAD65-GFP replenishing the Golgi in COS-7 cells. The Golgi-associated pool of palmitoylation-deficient (A) and wt GAD65-GFP (B) in living COS-7 cells were selectively photobleached (circle, t=0) and fluorescence recovery was monitored over time at 37°C. Note the presence of wt GAD65-GFP in cytosolic punctate structures, whereas the mutant protein is localized in the ER network of membranes. (C) Kinetics of fluorescence recovery after bleaching of the entire Golgi-associated pool of wild-type GAD65-GFP (red circles, n=10 cells) and GAD65(C30,45A)-GFP (blue circles, n=10 cells). Recovery of GFP fluorescence into the photobleached Golgi compartment was recorded at low laser power and normalized taking into consideration the loss of GFP fluorescence in the whole cell during the recording. Data are presented as mean ± s.d. from a representative experiment. (D) Half-times of Golgi fluorescence recovery following photobleaching for wild-type and palmitoylation-deficient GAD65-GFP. (E) Percentage recovery of Golgi fluorescence following photobleaching. Data for D and E are presented as mean ± s.e. from four independent experiments (5-10 cells per experiment).
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