Galectin-9 CRD2 is presented at the surface of NPC exosomes. Low-density vesicles (110-kg pellets) released by C17 or C666-1 NPC cells were incubated with magnetic beads coated with the 9M1-3 monoclonal antibody directed to the CRD2 of galectin-9. Beads coated with purified isotype-matched nonspecific Ig were used for control reactions. (Top panel) Numerous exosomes released by C17 (A) or C666-1 (B) are captured by anti-CRD2, but not control beads, and visualized by electron microscopy. (Middle panel) Western blot analysis detects the DR-α and galectin-9 proteins in C17 (C) and C666-1 (D) exosomes, whereas no similar proteins are recovered from control beads coated with irrelevant IgG. Positive controls are provided by total C17 and C666-1 cell extracts in the left lanes of panels C and D, respectively. (Bottom panel) A small amount of the galectin-9 CRD2 is detected at the surface of live C17 cells by flow cytometry (E), whereas it is absent at the surface of C666-1 (F) cells.