Anti cd63

The size distribution and concentration of the nanoparticles before and after treatment were characterized by NTA. Each NTA video was 60 s, and each result was averaged by at least three tests. The concentration of the preprocessed polydisperse sample in the NTA test was diluted to guarantee that there were 70 to 120 particles per frame to ensure statistics and suppress the overlapping effect. For monodisperse samples, at least 10 particles per frame were guaranteed.
For protein extraction and Western blot, plasma samples were lysed with lysis buffer and subjected to 10% SDS–polyacrylamide gel electrophoresis gels and transferred to a polyvinylidene difluoride membrane. Anti-CD63 (1:200; Santa Cruz Biotechnology) and anti-CD81 (1:200; Santa Cruz Biotechnology) antibodies were used as primary antibodies to confirm the presence of exosomes. The bound primary antibody was detected by peroxidase-conjugated goat anti-rabbit immunoglobulin G (Sigma-Aldrich) and the ECL Western blot analysis system (Millipore). Each experiment was performed at least three times.

Lysates of cells or exosomes were diluted at a ratio of 1:5 with protein loading buffer (5×; Beyotime Biotechnology, Jiangsu, China) and heated at 95 °C for 5 min. Protein extracts were separated by SDS-PAGE (6% gel for DMBT1 and 12% gel for other proteins) and transferred to polyvinylidene fluoride membranes (Immobilon P, Millipore, USA). The membranes were blocked with 5% milk in TBST (Tris-buffered saline, 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20) for 60 min at room temperature, incubated with primary antibodies at 4 °C overnight and then incubated with the horseradish peroxidase-conjugated secondary antibodies at 37 °C for 1 h. Primary antibodies and dilutions were used as follows: anti-CD9 (1:500; Santa Cruz), anti-CD63 (1:300; Santa Cruz), anti-CD81 (1:500; Santa Cruz), TSG101 (1:1000; ProteinTech, Chicago, USA), anti-DMBT1 (1:5000; Abcam, Cambridge, Britain), anti-VEGF-A (1:500; R&D system, Minneapolis, USA), Akt (1:500; Cell Signaling Technology, Danvers, USA), anti-phosphorylate Akt (p-Akt; 1:500; Cell Signaling Technology) and anti-GAPDH (1:5000; Cell Signaling Technology). All the secondary antibodies (1:5000) were obtained from Cell Signaling Technology. The immunoreactive bands were visualized using enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, USA) and imaged by the ChemiDoc XRS Plus luminescent image analyser (Bio-Rad).