Characterization and specificity of Ig binding to exosomes. A: Western blot of exosomes from mouse plasma isolated with ExoQuick, with a 20-μg protein loading volume. Exosomes isolated from mouse plasma showed enrichment of exosomal markers (CD63, ALIX, TSG101, CD9) than whole plasma. Specificity of exosome isolation was confirmed using lipoprotein markers (ApoE and ApoA) and the endoplasmic reticulum marker calnexin. B: Western blot of total circulating exosomes. Exosomes were separated with OptiPrep Density Gradient after ExoQuick isolation. The exosomal markers CD63, TSG101, and CD9, as well as Ig, are present in fractions with a density of 1.16–1.39 g/mL (fractions 8–10). C: Ig is removed in exosome-depleted plasma (right lane).