Fig. 3. Palmitoylation is required for targeting of GAD65-GFP to vesicles. (A-F) Projected confocal images of rat hippocampal neurons (A,B), MDCK cells (C,D) and COS-7 cells (E,F), transiently expressing either wt GAD65-GFP (A,C,E) or the palmitoylation-deficient mutant GAD65(C30,45A)-GFP (B,D,F). Rat hippocampal neurons were transfected at DIV6, fixed 72 hours following transfection and immunostained for GFP before confocal analyses. COS-7 and MDCK cells were fixed 18 hours post-transfection and analyzed by confocal microscopy. In addition to Golgi localization, GAD65-GFP-containing vesicles were observed in soma, dendrites (arrows) and axons (arrowheads) of rat hippocampal neurons and in the cytoplasm of fixed COS-7 and MDCK cells (see also supplementary material Movies 1 and 2). By contrast, palmitoylation-deficient GAD65-GFP was detected in Golgi membranes and in a diffuse pattern throughout the cell but not in vesicles in any of the three cell types (see also supplementary material Movie 3). (G) Quantification of wt GAD65-GFP and GAD65(C30,45A)-GFP positive vesicles in the cytosol of COS-7 cells. Results are presented as mean ± s.e. for five cells for each protein. ^*P<0.001.