(A) Immunoblot of FGFR1, FGF2 and actin in HS-5 and HS-27 whole cell lysates. (B) HS-5 and HS-27 CM were ultracentrifuged at 100,000 g for 2 hr at four degrees C. CM, soluble protein (S100), and ECV (P100) fractions were collected and analyzed by immunoblot, using 10, 50, and 100 ng/ml recombinant FGF2 for comparison. The ultracentrifuge tube was also washed with detergent to remove adherent ECVs and material (detergent wash P100). (C) HS-5 CM, S100 and P100 fractions (concentrated ~10 fold compared to HS-5 CM) were solubilized in 0.1% NP-40 and analyzed by cytokine multiplex ELISA (Luminex). The S100 and P100 fractions were normalized to CM. (D) The HS-5 P100 fraction (starting material, or SM) was further fractionated on a sucrose step-gradient. Sucrose layer interfaces (0–7.5%, 7.5–15%, 15–30%, 30–45%, and 45%-pellet) were collected, lysed and analyzed by immunoblot with antibodies against the exosomal marker CD9, FGF2, and cytoplasmic marker actin. (E) HS-5 and HS-27 ECVs (P100), recombinant FGF2, and HS-5 cells were exposed to proteinase K and analyzed by immunoblot. (F) HS-5 exosomes were isolated by sucrose step-gradient (see panel D) and then exposed to proteinase K with or without detergent (0.1% Triton X-100, used to dissolve the lipid membrane). Samples were subjected to immunoblot analysis using antibodies against tsg101, CD9 and FGF2. The * indicates degraded FGF2 after partial proteinase K digestion.