SDS-PAGE analysis of the EV proteins was performed by SDS-PAGE gel (Bio-Rad, Berkeley, CA) followed with silver staining using a ProteoSilver Plus Silver Stain Kit (Sigma). For EV fractions with different elution times, 6 and 14 min, respectively, the EV proteins derived from 40 μL of serum were loaded onto the gel, with 0.05 μg of BSA standard protein (Abcam, Cambridge, MA) employed as a reference. For the concentrates filtered by the three Amicon RC centrifugal filters (MWCO 10/50/100 kDa), the EV proteins from the same amount of 10 μg of EV standard were loaded onto the gel, with BSA again employed as a reference.
EV proteins (~5 μg) were separated by SDS-PAGE gel (Bio-Rad) and transferred to a PVDF membrane (Bio-Rad). The PVDF membranes were blocked for 1 h at room temperature in PBST containing 5% milk and incubated overnight at 4°C with the following primary antibodies in PBST containing 2% BSA: mouse anti-human CD9 monoclonal antibody (1:1000) (ab254175, Abcam, Cambridge, UK) and mouse anti-human CD63 monoclonal antibody (1:1000)(ab1318, Abcam). After incubation with appropriate HRP-conjugated secondary antibodies (1:3000) in PBST containing 2.5% milk, blots were performed using Super signal Chemiluminescence Reagent (Thermo Scientific, Rockford, IL).
EV proteins (~5 μg) were separated by SDS-PAGE gel (Bio-Rad) and transferred to a PVDF membrane (Bio-Rad). The PVDF membranes were blocked for 1 h at room temperature in PBST containing 5% milk and incubated overnight at 4°C with the following primary antibodies in PBST containing 2% BSA: mouse anti-human CD9 monoclonal antibody (1:1000) (ab254175, Abcam, Cambridge, UK) and mouse anti-human CD63 monoclonal antibody (1:1000)(ab1318, Abcam). After incubation with appropriate HRP-conjugated secondary antibodies (1:3000) in PBST containing 2.5% milk, blots were performed using Super signal Chemiluminescence Reagent (Thermo Scientific, Rockford, IL).