Western Blot Immunostaining of EV Proteins

Immunostaining of EV-derived proteins was performed by Western blotting as previously described [22 (link), 23 (link)]. Exosome samples were diluted 1 : 1 in Exosome Sample Buffer (5% SDS, 9 M urea, 10 mM EDTA, 120 mM Tris-HCl, pH 6.8, 2.5% beta-mercaptoethanol) and heated (95°C, 5 min). For cell samples, cells were scraped from the culture surface, resuspended in lysis buffer (1% SDS and 0.1% Triton X-100 in PBS with protease inhibitors (cOmplete Mini, EDTA free, Roche)), and incubated on ice for 30 min. Genomic DNA was sheared through a 27 G needle 4 times. Subsequently, membranes were incubated in either of the following antibodies: rabbit-anti-GAPDH (Cell Signaling, Boston, MA, USA), rabbit-anti-Flottilin-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat-anti-Lamin A/C (Santa Cruz Biotechnology), mouse-anti-ATP5A (Abcam, Cambridge, UK), or rabbit-anti-Tom20 (Santa Cruz Biotechnology).
As secondary antibodies, 1 : 2,000 diluted affinity-purified swine anti-rabbit, rabbit-anti-mouse, or donkey anti-goat coupled with horseradish peroxidase (Dako, Glostrup, Denmark) were used. Antigen-antibody reactions were visualized with enhanced chemiluminescence according to the manufacturer's guidelines (Chemiluminescent Peroxidase Substrate, Sigma) and imaged using a GelDoc XR+ system (Bio-Rad, Hercules, CA, USA).