After isolation, EV samples were resuspended in RIPA lysis buffer. RBC ghosts and human platelet lysates were prepared as in (Prausnitz et al., 1993 (link); Octave et al., 2021 (link)) respectively. Equal protein amounts (except for plasma samples and platelet lysates) were diluted in a buffer containing 10 mM of dithiothreitol (DTT) and then loaded for sodium dodecylsulfate polyacrylamide gel electrophoresis (Mini-Protean TGX Precast Gels 4%–15% (w/v) SDS-PAGE; BioRad or Novex 4–12% Tris-Glycine Gels, Invitrogen). Then, proteins were transferred to polyvinylidene fluoride (PVDF) membranes and blocked for 2 h. Membranes were incubated overnight with anti-apolipoprotein B100 (Apo B100; BioConnect, SC-13538; 1:500), anti-apolipoprotein A1 (Apo A1; BioConnect, SC-376818; 1:500), anti-CD41 (Abcam, ab134131; 1:2,000), anti-glycophorin A (GPA; Merck, MABF758; 1:1,000), anti-flotillin 1 (BD Biosciences, BD610820; 1:500), anti-ankyrin (Merck, MAB1683; 1:1,000), anti-spectrin (α and β) (Merck, S3396; 1:500), anti-stomatin (Abcam, ab67880; 1:500) or anti-band3 (Invitrogen, MA1-20211; 1:4,000) antibodies. Secondary peroxidase-conjugated goat anti-rabbit or anti-mouse IgGs were then incubated for 1 h and washed. Signal revelation was performed with SuperSignalTM West Pico or Femto (ThermoScientific).
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