Anti tsg101

Protein extracts were precleared with 50 % protein A sepharose (Amersham Biosciences), and incubated with 2 ug of anti-TSG101 (Novus) or -GFP (Millipore) antibodies overnight at 4°C. Thereafter, 100 μl of 50% protein A sepharose were added and incubated for an additional 2 h. The sepharose bead-bound immunocomplexes were washed three times with 1x PBS and dissolved in 2x SDS sample buffer.

Isolated EVs were re-suspended in Laemmli buffer and boiled for 5 minutes at 95 °C. Specifically, 20 µg of EVs were prepared in non-reducing conditions for tetraspanins detection, while 35 µg were used for soluble protein detection. Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Nonspecific sites were saturated with 5% (w/v) skimmed milk in Tris-buffered saline: 150 mM NaCl, 20 mM TrisHCl, pH 7.4, and 0.5% Tween 20 (T-TBS). Membranes were incubated overnight at 4 °C with anti-CD9 (1:1000, BD Pharmingen, #555370, San Jose, CA, USA), anti-CD63 (1:2000; BD Pharmingen, #556019, San Jose, CA, USA), anti-Alix (1:500, Santa Cruz, #sc-271975, Santa Cruz, CA, USA), anti-TSG101 (1:500, Novus Bio, #NB200–112, Littleton, CO, USA), anti-DCXR (1:1000, Abcam, #ab110283, Abcam Inc., Cambridge, UK), anti-Calnexin (1:1000, Sigma, #C7617, Sigma-Aldrich, St. Louis, MO, USA) and anti-β-tubulin (1:5000, Santa Cruz, #sc-9104, Santa Cruz, CA, USA). After washing with T-TBS, membranes were incubated with the horseradish peroxidase-conjugated secondary antibodies diluted 1:2000 for 45 min. Immunoreactive bands were revealed by the enhanced chemiluminescence method (ImmobilonHRP substrate, #WBKLS0500, Millipore Corp., Billerica, MA, USA).