Fig. 5. The impact of syntaxin 8 overexpression on GFP-CFTR cellular localization in COS-7 cells. (A-C) Thick optical sections acquired with 2, 0.6 or 0.8 µm steps (from top to bottom). (D-F) Projections of entire Z series. Cells were transfected with GFP-CFTR alone (A,D), cotransfected with GFP-CFTR and Syn8 (B,E) and cotransfected with GFP-CFTR and the cytosoluble form of syntaxin 8: Syn8TM (C,F). Green fluorescence resulting from GFP-CFTR expression shows strong and continuous localization of GFP-CFTR concentrated in plasma membrane of control cells (A,D). In doubly transfected cells, overexpression of Syn8 (as revealed by red fluorescence) is associated with a strong reduction of GFP-CFTR plasma membrane localization, while a partial colocalization is visualized in a juxtanuclear region as revealed by yellow fluorescence. By contrast, experiments using Syn8TM exhibit a large cytosolic staining of syntaxin 8 soluble form and a plasma membrane staining for GFP-CFTR (C,F). (G) Control experiments studying NCX2 localization in wild-type or Syn8-transfected COS-7 cells. Arrowheads indicate plasma membrane localization of NCX2 in both cases. Bars, 20 µm. Each image is representative of six independent experiments.