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Fig. 8. The slowly recovering pool of GAD65-GFP in Golgi membranes is dependent on palmitoylation in both neurons and COS-7 cells. FRAP analysis of the Golgi-associated pool of GAD65(C30,45A)-GFP and wild-type GAD65-GFP in hippocampal neurons (A-D) and COS-7 cells (E-H). Transfected cells were treated for 5 hours at 37°C with either 25 µM 2-BP or DMSO followed by selective photobleaching of the Golgi-associated pools. (A,B,E,F) Kinetics of recovery of GFP fluorescence for palmitoylation-deficient GAD65(C30,45A)-GFP (A,E) and wt GAD65-GFP (B,F). Data for neurons are presented as mean ± s.d. (n=10 cells for each condition). Data for COS-7 cells are presented as mean ± s.d. (n=5 cells for each condition). (C,G) Half-times of recovery of Golgi fluorescence following photobleaching of wild-type and palmitoylation-deficient GAD65-GFP in the presence and absence of 2-BP. The slow phase of GAD65-GFP recovery in Golgi membranes is abolished in the presence of 2-BP. (D,H) Percentage recovery of Golgi fluorescence following photobleaching. Data for C and D are presented as mean ± s.e. (n=10 neurons for each condition). Data for G and H are presented as mean ± s.e. (n=5 COS-7 cells for each condition).

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