Fig. 4. Palmitoylation results in spatially distinct localization of GAD65-GFP in Golgi membranes of rat hippocampal neurons and increased colocalization with the trans-Golgi marker TGN38. High-resolution projected confocal images of rat hippocampal neurons at DIV10 transiently expressing either palmitoylation-deficient (A,B) or wild-type (C,D) GAD65-GFP. Neurons were double immunolabeled for GFP and either the trans-Golgi network protein TGN38 (A,C) or cis-Golgi matrix protein GM130 (B,D). Palmitoylation-deficient GAD65 segregates from both TGN38 (A) and GM130 (B) and appears to be oriented more proximal to the cytosol than the two markers. Wild-type GAD65-GFP, however, colocalizes with TGN38 (C) but segregates from GM130 (D) albeit to a distinct face of GM130 than palmitoylation-deficient GAD65. GAD65-GFP-containing vesicles are observed for wt GAD65 (arrowheads) but not palmitoylation-deficient GAD65. (E) Correlation coefficient (r) for colocalization of palmitoylation-deficient and wt GAD65-GFP with TGN38. Only wt GAD65-GFP colocalizes significantly with TGN38 (r=0.80±0.02). Results are presented as mean ± s.d. for 10 cells for each protein. ^*P<0.001.