Western Blot Protein Detection Protocol

Western blot assay was performed as described previously (55 (link), 95 (link)). Briefly, cells or purified exosomes were lysed in NP-40 lysis buffer (10 mM Tris-HCl buffered at pH 7.5, 150 mM NaCl, 0.5% NP-40, 1% Triton X-100, 10% glycerol, 2 mM EDTA, 1 mM NaF, 1 mM Na₃VO₄ and 1% protease inhibitor mixture). The cell lysates were separated by centrifugation at 12,000 rpm for 10 min at 4°, and the concentration was measured by the Bradford method. The lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were further blocked with 5% non-fat dry milk at room temperature for 1 h, followed by sequentially incubating with primary antibodies and secondary antibodies. The antibodies-incubated membranes were visualized with Odyssey CLX Imager (LI-COR Biosciences) and analyzed by Image Studio Lite Ver 4.0 (LI-COR Biosciences).