FIGURE 3:

Cbl and Cbl-b are essential for sequestration of the CSF-1R and subsequent degradation within macropinosomes. (A) Immunofluorescence of the CSF-1R with and without inhibition of the vacuolar ATPase with 30 min pretreatment with bafilomycin A1 followed by CSF-1 exposure time course (images are identically window leveled and were collected under identical conditions). (B) Immunofluorescence staining of the intracellular and extracellular epitopes of the CSF-1R with selective permeabilization of the plasma membrane with digitonin or complete membrane permeabilization with Triton-x 100 at 15 min post CSF-1 stimulation. Gray arrows denote locations of macropinosomes. Green staining in during digitonin indicates cytosolic accessibility of the CSF-1R. (C) Distribution of LAMP1-positive lysosomes in relation to the CSF-1R contained with the lumen (WT) or limiting membrane of the macropinosome (DKO) at 15 min post CSF-1 stimulation. (D) Quantification of the CSF-1R localization from 30 macropinosomes for each WT and DKO based on morphology in the Triton-X 100 staining condition.