Galectin-5 is secreted through the exosomal pathway. Exosomes were collected by differential centrifugation from the culture medium of in vitro reticulocyte maturation in various conditions. (A) Increasing amounts of exosomes (10-80 μg of protein) were processed by SDS-PAGE and analyzed by Western blot for the presence of TfR and Gal-5 by the use of appropriate antibodies. (B) Exosomes released during 2, 6, and 24 hours of reticulocyte (100 μL of packed cell volume) in vitro maturation were isolated from the culture medium and compared for their TfR and Gal-5 content. (C) Exosomes collected from the medium after in vitro maturation (24 hours, 37°C) of reticulocytes in the absence (Ctrl) or presence of sodium fluoride (20mM), chloroquine (10μM), monensin (1μM), or after reticulocyte maturation at 4°C were analyzed.