Ez western detection kit

Exosome pellets were suspended with 1× PBS and lysed with RIPA buffer containing protease and phosphatase inhibitors. Proteins were diluted with 5× sample buffer, separated by SDS-PAGE, and transferred to a nitrocellulose membrane. The membranes were blocked with PBS containing 0.1% Tween-20 and 5% skim milk and then incubated with primary antibodies at 4 °C overnight. The blots were incubated with horseradish peroxidase-conjugated secondary antibodies and detected using an EZ-western detection kit (Dogen-Bio, Seoul, Korea). Anti-Alix, -CD9, and -GAPDH antibodies were purchased from Cell Signaling (Danvers, MA, USA).

Total protein was extracted with RIPA buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% triton X-100, 2 mM EDTA, 0.1% SDS, and 1% sodium deoxycholate. Protein concentration was quantified by Bradford assay, and proteins were separated on 8-12% SDS-PAGE. Separated proteins were transferred to nitrocellulose membrane and blocked with 5% skim milk in PBS-T at room temperature for 1 h. The membrane was incubated with primary antibodies at 4°C overnight, washed, and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. Proteins were measured using the EZ-western detection kit (Dogen-Bio, Seoul, Republic of Korea).