Extracellular Vesicle Protein Characterization

Extracellular vesicle containing fractions were lysed in 1X RIPA. Protein concentrations were determined by microBCA protein assay (Thermo Fisher, 23235). Equivalent protein amounts (extracellular vesicles and proteins) were separated on 4–15% stain-free precast SDS-PAGE gradient gels (Bio-Rad 5678083) under nonreducing conditions and transferred onto PVDF membranes (Sigma Aldrich IPVH00005). After 1 h blocking (5% nonfat milk, Bio-Rad 170–6404) at room temperature (RT), membranes were incubated with antibodies against CD63 (1 : 1000, BD Biosciences 556019), CD81 (1 : 500, Santa Cruz Biotechnology sc23962), calnexin (1 : 2000, Abcam ab22595), GM130 (1 : 1000, Abcam, ab76154), albumin (1 : 1000, Abcam ab28405), AGO2 (1 : 500, Sigma-Aldrich SAB4200085), ApoB100 (1 : 1000, Academy Bio-Medical 20A-G1b), ApoA1 (1 : 1000, Academy Bio-Medical 11A-G2b), and ApoC1 (1 : 1000, Academy Bio-Medical 31A-G1b) overnight at 4°C. Membranes were washed three times for 8 min in PBST with shaking, then incubated with HRP-conjugated secondary mouse antirabbit IgG or mouse IgG kappa binding protein antibodies (1 : 10 000, Santa Cruz Biotechnology sc-2357 and sc-516102) at RT for 1 h. After a PBST wash, membranes were incubated with SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher 34580) and visualized by iBright (Thermo Fisher, Waltham, Massachusetts, USA).