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Fig. 9. GAD65-GFP accumulates in the ER and Golgi and is absent from cytosolic vesicles in the presence of 2-BP. (A-C) High-resolution projected confocal images of COS-7 cells expressing the ER-marker DsRed2-ER together with either wild-type (A,B) or palmitoylation-deficient (C) GAD65-GFP. Transfected COS-7 cells were allowed to express the protein for 16 hours and then treated with 25 µM 2-BP (B) or DMSO (A,C) for 5 hours at 37°C before fixation and immunofluorescence analysis. Wild-type GAD65-GFP is present in cytosolic vesicles and Golgi in the absence of 2-BP and does not colocalize with DsRed2-ER (A, enlarged frames a and b). However, inhibition of palmitoylation results in the disappearance of wt GAD65-GFP from cytosolic vesicles and accumulation in ER membranes (B, enlarged frames c and d), a localization similar to that shown for the palmitoylation-deficient GAD65-GFP (C, enlarged frames e and f). (D) Correlation coefficient for colocalization of wt GAD65-GFP with the DsRed2-ER marker following incubation with a solvent (DMSO) and 2-BP, respectively. Results are presented as mean ± s.e. (n=10 cells per condition). ^*P<0.001. (E) Quantification of wt GAD65-GFP positive vesicles in the cytosol of COS-7 cells treated with solvent (DMSO) and 2-BP. Results are presented as mean ± s.e. (n=5 cells per condition). ^*P<0.001.

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