IgM antibodies bind to exosomes. (A) Exosomes were obtained by differential centrifugation and surface-associated proteins were released by a carbonate wash. Untreated exosomes, stripped vesicles, and the released proteins were loaded on SDS-PAGE and analyzed by Western blot for IgM and then for TfR. The membrane was then reprobed for flotillin. (B) Exosomes were deposited on a linear sucrose gradient. Fractions were collected and analyzed for the presence of IgM (top panel) and TfR (bottom panel) using specific antibodies. Proteins stripped from exosomes by carbonate wash were deposited on a sucrose gradient and analyzed for IgM (middle panel). Densities (g/mL) were obtained for each fraction by refractometry and are indicated under each lane. (C) Proteins released from exosomes following a carbonate wash (released proteins) were added back to stripped vesicles (1 h at room temperature) and submitted to ultracentrifugation (200 000g for 10 minutes). Resulting pellets and supernatants were then analyzed for the presence of IgM and hsc70. As controls, stripped vesicles and carbonate wash were submitted to the same centrifugation conditions separately, as indicated at the bottom, and analyzed by Western blot. The molecular mass (kDa) standards are indicated to the left. Arrows point to the bands corresponding to Western blot detection of specified proteins.