Determining Exosomal THBS2 Protein Localization

To determine the location (membrane or inside of exosomes) of exosomal THBS2 protein, Proteinase K assay was performed, as described previously 34 (link). 20 mL plasma of 6 LUAD patients were used to purify exosomes (500 μL; concentration: 1062 ng/μL), and were incubated in (1) PBS (control), (2) 1.2 μg/mL Proteinase K (Promega, Catalog Number: V3021; in PBS) alone, (3) 0.5% Triton X-100, or (4) combined Proteinase K and Triton X-100. The samples were then subjected to immunoblot to test TSG-101 (exosomal intra-membrane protein; as positive control), CD9 (exosomal trans-membrane protein; as positive control), THBS2.