FIGURE 3.

EVs derived from human serum contain TTR, and purified TTR binds to the serum EVs. (A) Analysis of EVs derived from serum (S-EVs) by NanoSight (Raw mode). The particle concentration was 4.15 × 10¹⁰ ± 3.30 × 10⁹. (B) Multiple molecular weight TTR and TTR aggregates were detected in western blotting analysis of serum and S-EVs. (C) Mixture of S-EVs or Elution Buffer and purified TTR in static conditions for 48 h used for western blotting analysis. V30M-TTR with or without S-EVs exhibited ladder bands. (D) Remaining samples were used for EV ELISA. S-EVs with V30M-TTR showed the highest absorbance. S-EVs with WT-TTR also exhibited an increase in absorbance, whereas TTR without EVs showed little increase in absorbance. N = 3, mean ± S.E.; **, p < 0.005; n.s., not significant, ANOVA with Tukey’s post hoc test. (E) S-EVs in PBS were imaged with HS-AFM. EV particles were observed fixed to the substrate. Particles were mainly smaller than those confirmed by Nanosight. (F) Change in S-EV particle height over time under five conditions. Height of serum EVs increased in a time-dependent and TTR-concentration-dependent manner. Acid buffer (Acid). All AFM movies were taken at 250 ms/frame. Scanning area was 100 × 100 nm² with 80 × 80 pixels. (G) Time course of 2D correlation coefficients of the surface of EVs under the above five conditions. The 2D correlation coefficient calculation has been described in the Materials and Methods “Analysis of HS-AFM Images” section in detail. The addition of TTR and an increase in TTR concentration reduced amplitude of the EV surface.