The obtained sEVs and lEVs from plasma were resuspended in 0.1% (v/v) Triton lysis buffer (Solarbio, China), and the sample loading amount normalized using bicinchoninic acid protein quantification (Solarbio). Proteins (10 μg) were separated using 10% sodium SDS-PAGE. The separated proteins were transferred onto PVDF membranes blocked with 5% (w/v) non-fat dry milk and incubated at room temperature (RT) for 1 h. The membranes were washed and incubated overnight at 4 °C with primary antibodies against CD63 (1:1000, ab92726, Abcam, UK), HSP70 (1:1000, 66,183-1-IgM, Proteintech, China), Apo-A1 (1:5000, 14,427-1-AP, Proteintech), and Grp94 (1:2000, 60,012-2-Ig, Proteintech). The following day, the membranes were washed and incubated at RT for 1 h with secondary antibodies (goat anti-rabbit/mouse IgG H&L chain conjugated to HRP, 1:2000, Abcam). All washes comprised 10 min washes with 0.05% (v/v) Tween-20 (Solarbio) at least three times. The antigen–antibody binding complexes were detected using an enhanced chemiluminescence reagent (Thermo, USA), and the bands were visualized using a chemiluminescence imager (Tanon, China).
For coomassie brilliant blue (CBB) staining, the proteins (10 μg) were separated as before using SDS-PAGE, and the resulting gels were immersed in the Coomassie dye solution (BeyoBlue, China) for 2 h at RT and eluted overnight with ultrapure water.
For coomassie brilliant blue (CBB) staining, the proteins (10 μg) were separated as before using SDS-PAGE, and the resulting gels were immersed in the Coomassie dye solution (BeyoBlue, China) for 2 h at RT and eluted overnight with ultrapure water.