Shi exo m01

NSCLC patient‐derived plasma samples (200 μl) were fractionated on an EVSecond L70 column according to the manufacturer's instructions. For fraction check analysis, each fraction (100 μl) was dried in a vacuum drier and resuspended in 20 μl Laemmli's SDS sample buffer. To acquire whole exosome‐containing fractions, the second to fifth fractions were combined and subjected to acetone precipitation before resuspension in Laemmli's SDS sample buffer. For analysis of whole cell lysates, cells were lysed with Laemmli's SDS sample buffer at a concentration of 1 mg/ml. Next, 10 μg each of protein sample was separated on 8%–12.5% SDS polyacrylamide gels and transferred onto PVDF membranes (Merck Millipore, #IPVH00010). Following blocking with 4% Block Ace (Yukijirushi Nyugyo, Tokyo, Japan), membranes were incubated with anti‐DOK3 polyclonal antibody (HPA077312, Sigma‐Aldrich), anti‐CD9 monoclonal antibody (SHI‐EXO‐M01, Cosmo Bio), or anti‐calnexin polyclonal antibody (ab22595, abcam). Membranes were then incubated with HRP‐conjugated anti‐mouse IgG (GE Healthcare, Chicago, IL, #NA931‐1Ml) or anti‐rabbit IgG (GE Healthcare, #NA934‐1Ml) and detected using Western Lightning ECL Pro (Perkin Elmer, MA, #NEL121001EA). Quantification of band intensity was performed using Image Lab software version 5.2 (Bio‐Rad Laboratories,).