Galectin-5 is located on the exosome surface. (A) Exosomes (400 μg of protein; bottom and middle) or purified Gal-5 (3 μg; top) were carefully loaded on a linear sucrose gradient. Fractions were collected from the top of the gradient, processed by SDS-PAGE, and analyzed by Western blot for the presence of TfR (bottom) and Gal-5 (middle and top) by the use of specific antibodies. Densities (g/mL) were obtained for each fraction by refractometry. (B) Exosome surface–associated proteins were released by a carbonate wash. Untreated exosomes and stripped vesicles were processed by SDS-PAGE and analyzed by Western blot for TfR and Gal-5. (C) Exosome material was coated on latex beads and analyzed by flow cytometry for Gal-5 by the use of anti–galectin-5 serum (solid line) or a preimmune serum (tinted pattern) and Alexa Fluor 488 donkey anti–rabbit IgG (left) or by the use of purified anti–galectin-5 IgGs and Alexa Fluor 488 donkey anti–rabbit IgG (solid line; right). As a control BSA-coated beads (dashed line) were treated similarly. Tinted patterns indicate the absence of primary antibody on exosome-coated beads. (D) Exosomes preincubated with purified anti–galectin-5 antibody (2 μg, 1 hour, RT; top and middle) or Gal-5 antibody (2 μg; bottom) were loaded on a linear sucrose gradient. Fractions were collected and analyzed for the presence of TfR (top) and Gal-5 antibody (middle and bottom) by the use of specific antibodies.