Tsg101

SDS-PAGE was used to separate equal amounts of EV protein (quantified by Sypro^® Ruby staining). Proteins were transferred to the nitrocellulose membrane (Thermo Scientific, Waltham, MA, USA), blocked with 5% skim milk and probed with the following antibodies: TSG101 (BD Transduction Laboratories: catalogue number—612696) and Alix (Cell Signaling Technology, Danvers, MA, USA: catalogue number—2171S). Fluorescent conjugated rabbit and mouse secondary antibodies were used, and the protein bands were visualized using ODYSSEY CLx (LI-COR^®).

SDS-PAGE was used to separate equal amounts of protein (10 or 30 μg quantified by Sypro^® Ruby stain) from EVs and WCL samples. Gels were transferred to nitrocellulose membrane using an iBlot™ gel transfer stack system (Life Technologies). Membranes were then blocked with skim milk and probed overnight with the primary antibodies against TSG101 (BD Transduction Laboratories), Alix (Cell Signaling), GM130 (BD Transduction Laboratories), Caspase-3 (Cell Signaling) and Poly (ADP-ribose) polymerase-1/PARP-1 (Santa Cruz Biotech). Fluorescent conjugated rabbit and mouse secondary antibodies were used and the protein bands were visualized using ODYSSEY CLx (LI-COR^®).

Cells were washed (ice cold PBS) and lysed on ice with SDS sample buffer (4% SDS, 20% glycerol, 0.01% bromophenol blue, 0.125 M Tris-HCl, pH 6.8. Lysates were subjected to ultracentrifugation for 30 min (336,000 × g, TLA-100 rotor, Beckman Coulter), and soluble supernatants retained for downstream use, or frozen at −80°C. Protein quantification was performed as previously described [70 (link)].
For immunoblotting, membranes were probed with primary antibodies for 1 h in TTBS (50 mM Tris, 150 mM NaCl, 0.05% Tween 20) followed by appropriate secondary Abs coupled to horseradish peroxidase. Primary Abs for phospho-ERM (1:1000), Alix (1:1000) and N-cadherin (1:1000) and TGN46 (1:1000) were from Cell Signaling; for ezrin (1:5000) and β-actin (1:10000) from Sigma Aldrich; for CD63 (1:1000) and GFP (1:5000) from Calbiochem; for PS1 (1:1000) from Abcam; for TSG101 (1:1000) and flotillin 1 (1:1000) from BD Transduction Laboratories; for CD9 (1:1000) and annexin A7 (N-19, 1:1000) from Santa Cruz Biotechnology; for GAPDH (1:1000) from Merck Millipore; for PDPN from Acris Antibodies (NZ1, 1:1000). For E-cadherin and CD44 detection, the monoclonal Abs ECCD2 and HP2/9 (a generous gift of Dr. F. Sánchez-Madrid), respectively, were used at 1:1000 dilution. Peroxidase activity was developed using an enhanced chemiluminiscence kit as indicated by the manufacturer (Pierce).