Fig. 4

Validation of mass spectrometry identified proteins using Western blot and flow cytometry. a The presence of iNOS, TSG101 and S100A8 were validated with Western blot in pool A and pool B of healthy subjects and a fifth subject (“indep. donor”). The absence of calnexin in exosomes was also demonstrated. b CD14, CD9 and CD63 were validated by binding the exosomes to anti-MHC class II beads and analysis with flow cytometry. Grey filled curve shows isotype control and black open curve shows CD14, CD9 and CD63 respectively. c The iNOS enzyme was demonstrated to be biochemically functional by an activity assay. Nitric oxide synthase coverts l-arginine into nitric oxide, which is further converted to nitrite. The assay measures the concentration of nitrite (µM), shown on the y-axis. Control samples (“Ctrl”) show the baseline concentration of nitrite in nasal exosomes. Nasal lavage exosomes (“NLF exo”) samples shows a higher concentration of nitrite after the assay