Anti flotillin 2

Exosome and cell lysates were prepared by mixing in lysis buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 1% Triton X-100, 10% glycerol). Lysates were diluted four-fold with 4X Laemmli sample buffer, heated to 65°C for 5 min and separated on 4–20% acrylamide Tris-Glycine gradient gels (Life Technologies). Proteins were transferred to polyvinylidene difluoride membranes (EMD Millipore, Darmstadt, Germany), blocked with 5% bovine serum albumin in TBST and incubated overnight with primary antibodies. Blots were then washed with TBST, incubated with anti-Rabbit or anti-Mouse secondary antibodies (GE Healthcare Life Sciences, Pittsbugh, PA) and detected with ECL-2 reagent (Thermo Fisher Scientific). Primary antibodies used in this study were anti-YBX1 (Cell Signaling Technology, Danvers, MA), anti-GAPDH (Santa Cruz Biotechnology), anti-TSG101 (Genetex, Irvine, CA), anti-CD9 (Santa Cruz Biotechnology), anti-Flotillin 2 (Abcam, Cambridge, MA), anti-Alix (Santa Cruz Biotechnology) and anti-Ago2 (Cell Signaling Technology). For quantitative immunoblotting (in Figure 6), the same procedures were used, but were instead imaged using the LiCOR Odyssey imaging system.