Apical–basal gradient of endosomal GFP/Cherry-tagged Notch. (A) A schematic representation of the distribution of the NiGFP4Cherry5 fluorescence along the apical–basal axis in notum epithelial cells. The GFP fluorescence of NiGFP4Cherry5 is strong at the apical plasma membrane (top, green) and in early/sorting endosomes and weak in late basal endosomes (bottom, red) as a result of quenching. Conversely, the Cherry fluorescence is weak at the plasma membrane and at early apical endosomes as a result of slow maturation time and strong in late basal endosomes. (right) This results in opposite gradients of GFP and Cherry fluorescence along the apical–basal axis. (B) The GFP/Cherry mean intensity ratios measured in endosomes were plotted along the apical–basal axis of notum epithelial cells (apical, z = 0; n = 4 nota; GFP- and Cherry-positive endosomes were combined to calculate mean ratio values). Ratios were calculated from raw pixel intensity values in the GFP and Cherry channels without normalization.