Hpa077312

Paraffin‐embedded specimens were prepared from patients who showed a high or low level of plasma exosomal DOK3 (Exo‐DOK3). The slides were treated with xylene and ethanol to remove the paraffin. The ENVISION+ Kit/HRP (DakoCytomation,) was used to detect DOK3, CD19, and CD8. After blocking the endogenous peroxidase activity and nonspecific protein staining, affinity‐purified rabbit anti‐DOK3 polyclonal antibody (HPA077312; Sigma‐Aldrich), mouse anti‐CD19 monoclonal antibody (BT51E; Leica Biosystems), or mouse anti‐CD8 monoclonal antibody (NCL‐L‐CD8‐4B11; Leica Biosystems) were added as the primary antibody. Then, slides were treated with horseradish peroxidase (HRP)‐labeled anti‐rabbit IgG for DOK3 staining or anti‐mouse IgG for CD19 and CD8 staining, respectively. Finally, chromogen 3,3′‐diaminobenzidine (DAB) was added as the substrate. Serial sections of the tissue specimens were counterstained with H&E. Quantification of cells with positive staining was performed using ImageJ software. The “Brightness” of “Threshold Color” was set from 111 to 170 to defined DAB‐stained cells for all slides. Then the area of the defined DAB‐stained cells was measured within ~1.7 mm² of tissue sections. Finally, the positive area per unit tissue section (μm²/mm²) was calculated and shown.

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,).