EV pellets were homogenized in RIPA buffer containing 1% SDS, and protein estimated using the BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Western blotting was performed on the various tissue-specific EV as described previously.31 (link), 78 (link), 79 Briefly, 20-40 μg protein was loaded onto NuPAGE 4–12% Bis-Tris gels (Invitrogen) and run either under non-reducing (CD63 and CD81) or reducing (Hsp-70 and flotillin-1) conditions followed by their transfer onto nitrocellulose membranes using the iBlot 2 Gel Transfer Device (Invitrogen). Membranes were blocked in TBS SuperBlock buffer (Thermo Fisher Scientific) for 30 min, and immunoblotting carried out overnight at 4 °C with primary antibodies (see Table 1 for details). The next day, membranes were incubated with respective HRP conjugated secondary antibodies for 1.5 hr at room temperature on a rocker. Blots were developed with 1:1 solution of Radiance Chemiluminescent Substrate and Luminol/Enhancer (Azure Biosystems) and visualized using a c300 imaging system (Azure Biosystems). Images were quantified using the ImageJ software.