Time course of cargo distribution in EEA1- and APPL1-positive structures. Fluorescently labeled Tf and EGF were internalized for 30 s and chased for the indicated periods of time (A). All experimental data are the mean of four independent experiments. The intensity of Tf and EGF colocalized to EEA1 and APPL1 was corrected for apparent (random) colocalization (Kalaidzidis et al., 2015). (B and C) Kinetics of fluorescence intensity of Tf (B) or EGF (C) colocalized with EEA1-positive and APPL1-negative endosomes (red), APPL1-positive and EEA1-negative endosomes (blue), with double EEA1+APPL1–positive endosomes (green) and colocalized with none of them (black). The inset shows the part of the curve corresponding to the initial 3 min of the time course. (D) Time course of colocalization of EGF-with-Tf (magenta) and Tf-with-EGF (green) on all endosomes. (E) Time course of colocalization of EGF-with-Tf (magenta) and Tf-with-EGF (green) on APPL1-positive and EEA1-negative endosomes. This colocalization is defined as the ratio of the amount of EGF colocalized with Tf on APPL1 endosomes to the total amount of EGF on APPL endosomes. The same applies to Tf-with-EGF colocalization on APPL endosomes. Error bars represent SEMs.