Ab67880

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).

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).