Molecular imager versadoc 4000 system

Western blotting was used to identify the U87-MG exosome markers Alix, Tsg101, CD9 (30 (link)), and Bcl-2 as well as Bax, and caspase-3 protein expression in HUVECs. Cells and exosome pellets were lysed in lysis buffer (Roche Diagnostics, Mannheim, Germany). Subsequently, 5X protein loading buffer was added directly to the protein sample, and the sample was heated at 95°C for 5 min. Next, equal amounts of protein were loaded and separated on SDS-PAGE polyacrylamide gels, and proteins were blotted onto a nitrocellulose membrane (Whatman, Maidstone, Kent, UK). Primary antibodies included Alix (mouse monoclonal anti-Alix, 1:1,000), Tsg101 (mouse monoclonal anti-Tsg101, 1:200), CD9 (rabbit monoclonal anti-CD9, 1:1,000), Bax (rabbit monoclonal anti-Bax, 1:2,000), Bcl-2 (rabbit monoclonal anti-Bcl-2, 1:1,000), and caspase-3 (mouse monoclonal anti-caspase-3, 1:500) (all from Abcam, Cambridge, UK). The primary antibodies were incubated overnight at 4°C, followed by washing and the application of HRP-conjugated goat anti-rabbit secondary antibodies (1:2,000) and goat anti-mouse secondary antibodies (1:2,000) (both from Thermo Fisher). Proteins were detected via enhanced chemiluminescence (Thermo Fisher) and imaged with a Molecular Imager VersaDoc 4000 system (Bio-Rad).