Immunoblotting for Extracellular Vesicle Protein Analysis

Detection of sEV proteins was performed by immunoblotting as previously described (van Balkom et al., 2015 (link)). For immunoblotting, sEV and cell 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 27G needle four times. SDS‐PAGE, protein transfer and blocking were performed as described (van Balkom et al., 2015 (link)). Subsequently, membranes were incubated in either of the following antibodies: rabbit‐anti‐GAPDH (Cell Signaling), rabbit‐anti‐Flotillin‐1 (Santa Cruz Biotechnology), goat‐anti‐Lamin A/C (Santa Cruz Biotechnology), mouse‐anti‐ATP5A (Abcam), or rabbit‐anti‐Tom20 (Santa Cruz Biotechnology; Table 1).
As secondary antibodies 1:2000 diluted affinity‐purified swine‐anti‐rabbit, rabbit‐anti‐mouse, or donkey‐anti‐goat coupled to horseradish peroxidase (Dako) were used. Antigen‐antibody reactions were visualised with enhanced chemiluminescence according to manufacturer's guidelines (Chemiluminescent Peoxidase Substrate, Sigma) and imaged using a GelDoc XR+ system (Bio‐Rad).