Figure 2. AGJ vesicles robustly colocalize with lysosomal and autophagosomal markers but only insignificantly with endosomal markers. (A) Schematic representation of phago-/lysosomal and endo-/lysosomal cellular degradation pathways and respective marker proteins (highlighted with blue shading) that have been investigated in this study. (B) Cx43-GFP or Cx43-mApple expressing HeLa cells were either labeled with antibodies specific for endosomal and lysosomal marker proteins (a–d), cotransfected with LAMP-1-cerulean (e), or stained with the acidophilic fluorescent probe, LysoTracker Red (f). Representative merged confocal fluorescence images obtained 20–24 h after transfection are shown. Individual and merged fluorescence signals of the boxed areas are shown at higher magnification on the right. Robust colocalization of vesicular Cx43 was observed with lysosomal markers (LAMP-1, LAMP-1-cerulean, and LysoTracker, panels d-f, as indicated by yellow, the resulting color of overlaying red and green emission signals), but only insignificantly with endosomal markers (EEA1 and Rab7, panels b and c). A notable colocalization of Cx43-GFP was also observed with the GTP-binding protein Rab5 (panel a), described to be involved in early steps of endocytosis (uncoating of clathrin-coated vesicles), and in targeting cytoplasmic cargo to autophagic degradation (see text). Representative colocalizing vesicles are marked with arrows; GJs are marked with arrowheads.