Fig. 7. GFP-CFTR and syntaxin 8 colocalize at least in the recycling endosome complexes in doubly transfected COS-7 cells. In each experiment (except D), COS-7 cells were cotransfected with GFP-CFTR and Syn8, then costained with anti-syntaxin 8 antibody and anti-Lamp-1 (A), anti-TfR (B) or anti-Rab11 (C) antibodies. All cell images present the projection of the entire Z series sections acquired by fluorescent confocal microscopy. Fluorograms were obtained as described in Materials and Methods. In each experiment, protein colocalizations were analyzed with three fluorograms. Syntaxin 8 (a in A-C) and GFP-CFTR (b in A-C) exhibit perinuclear staining as expected. These two proteins colocalize in the perinuclear region as shown in the merged images (d in A-C) and fluorograms (e in A-C). Lamp-1 immunostaining pattern (Ac) exhibits no colocalization with GFP-CFTR and syntaxin 8 both on merged image (Ad) and fluorograms (Af,g), whereas the immunostaining profile obtained with TfR (Bc) presents a partial colocalization on merged picture (Bd) and fluorograms (Bf,g). To further analyze colocalization compartments, we selected plots with high fluorescence intensity and placed them on the fluorogram bisector line (square in Bf,g) to generate images showing the colocalization patterns between CFTR and TfR (Bf') or Syn8 and TfR (Bg'). The vesicular pattern obtained in each case is very similar and shows a colocalization between the three proteins within these vesicles. By contrast, Rab11 immunostaining (Cc) closely matches in the perinuclear region with CFTR and syntaxin 8 stainings (Cd). This high degree of colocalization is sustained by fluorogram data (Cf,g) displaying Rab11/CFTR or Rab11/Syn8 fluorescent plots placed on the bisector line. As a control, we have studied endogenous localization between Rab11 and Lamp-1 or between Rab11 and TfR in wild type COS-7 cells. As expected, no overlap was found between Lamp-1 and Rab11 stainings (Da), whereas a partial colocalization was observed in the perinuclear region between Rab11 and TfR (Db). Bars, 20 µm. Each analysis is representative of three independent experiments.