Western Blot Analysis of Extracellular Vesicles

All samples were lysed directly in 1.2X Laemmli sample buffer containing 5% BME and boiled for 5 min. Laemmli sample buffer recipe: 4% SDS (10% (w/v), 20% glycerol, 120 mM 1 M Tris–Cl (pH 6.8), and 0.02% (w/v) bromophenol blue in water. Sympathetic cultures were washed with PBS and lysed directly on the plate with 200 μL of 1.2X Laemmli sample buffer. P20 and P100 fractions were lysed directly in micro/ultracentrifuge tubes with 30 μL of 1.2X Laemmli. The sample buffer was pipetted up and down 50 times along the walls of the tubes to collect the entire pellet. Samples were run on 4–12% polyacrylamide gels with 7 μL of cell pellet fractions and 15 μL of P20 and P100 fractions loaded per well. Protein gels were transferred to nitrocellulose membranes using the Trans-blot turbo, blocked in 5% milk for 1 h, and incubated in primary antibody (Alix 1:1000, CD63 1:1000, CD81 1:1000, Cytochrome C 1:5000, Calreticulin 1:4000) diluted in 5% milk 0.1% TBST overnight at 4 °C on a rocker. Membranes were then washed 3 × with 0.1% TBST and secondary antibodies (1:20,000) diluted in 0.1% TBST were incubated for 1 h at room temperature. Blots were imaged using the Odyssey CLx imager and exposure was determined automatically by the software.