Intact exosomes protect galectin-9 against trypsin digestion. (A) Western blot detection of DR-α, CD63, and galectin-9 in exosomes released by C17 cells and purified on a sucrose gradient. Gp96 is a cytoplasmic protein typically absent from exosomes. (Left side) C17 total extract. (Right side) Exosome proteins. (B) Galectin-9 contained in C17 exosomes is quantified in a Western blot analysis by comparison to a series of recombinant galectin-9 samples ranging from 1 to 25 ng (rec gal-9). On average, 2 to 5 ng galectin-9 are contained in 50 μg exosome proteins. (C) Trypsin digestion assay, the same amount of C17 exosomes was used for each condition (35 μg total exosome proteins). They were subjected either to Triton lysis or mock treatment before incubation with trypsin. Two different trypsin concentrations, 15 μg/mL (high trypsin) or 0.01 μg/mL (low trypsin), were used in distinct experiments. For samples treated with low amounts of trypsin, short and long exposures of the blotted membrane are presented. Controls are displayed in lane 1 (no Triton lysis, no trypsin addition, and incubation at 4°C), lane 5 (no Triton, no trypsin, 37°C), and lane 9 (Triton lysis without trypsin addition and incubation at 37°C). Triton lysis of exosomes greatly enhances trypsin digestion of exosome-bound galectin-9 as shown in lanes 6, 7, and 8 (Triton lysis followed by trypsin digestion) as compared with lanes 2, 3, and 4 (mock treatment followed by trypsin digestion). After Triton lysis, undigested galectin-9 remained detectable only for the shortest time of incubation (5 minutes) with the smaller concentration of trypsin (0.01 μg/mL; lane 6). In lane 9, a substantial decrease of galectin-9 is observed despite the absence of trypsin, suggesting an effect of endogenous proteolytic enzymes after Triton lysis.