The following antibodies were used: mouse anti-Hsp70 (SC-24; Santa Cruz, Heidelberg, Germany, 1:1,000), rabbit anti-Flotillin-1 (F1180; Sigma–Aldrich, Taufkirchen, Germany, 1:1,000), mouse anti-Tsg101 (4A10; GeneTex, Irvine, CA, USA), mouse anti-Integrin αIIb (SC-59923; Santa Cruz, 1:1,000) and HRP-coupled secondary antibodies (Goat-anti-Mouse-HRP, 115-035-003, 1:10,000; Goat-anti-Rabbit-HRP, 111-035-003, 1:10,000; Dianova, Hamburg, Germany).
EV pellets dissolved in sample buffer were subjected to SDS–PAGE (4–12% Bis-Tris gel) and Western blotting (NuPAGE; Life Technologies, Darmstadt, Germany). For analysis of 10,000×g and 100,000×g pellets, 20 µl was loaded on the gel. Proteins were blotted onto a PVDF membrane. Next, the membrane was blocked with 4% milk powder, 0.1% Tween in PBS and incubated sequentially with primary and HRP-coupled secondary antibodies. Proteins were detected with chemiluminescence reagents (Luminata Crescendo, Merck Millipore, Darmstadt, Germany) and X-ray films. X-ray films were scanned and signal intensities were measured using ImageJ 1.44 h (National Institutes of Health, Bethesda, MD, USA).
EV pellets dissolved in sample buffer were subjected to SDS–PAGE (4–12% Bis-Tris gel) and Western blotting (NuPAGE; Life Technologies, Darmstadt, Germany). For analysis of 10,000×g and 100,000×g pellets, 20 µl was loaded on the gel. Proteins were blotted onto a PVDF membrane. Next, the membrane was blocked with 4% milk powder, 0.1% Tween in PBS and incubated sequentially with primary and HRP-coupled secondary antibodies. Proteins were detected with chemiluminescence reagents (Luminata Crescendo, Merck Millipore, Darmstadt, Germany) and X-ray films. X-ray films were scanned and signal intensities were measured using ImageJ 1.44 h (National Institutes of Health, Bethesda, MD, USA).