Primary antibodies against tsg101

HASC-derived EVs were lysed in RIPA buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.5% Triton X-100, 1% Na-deoxycholate, 0.1% sodium dodecyl sulphate and protease inhibitor cocktail). A total of 20 µg of protein was electrophoresed in a 10% SDS-PAGE gel and transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% BSA in T-TBS (10 mM Tris, 150 mM NaCl and 0.1% Tween 20) for 2 h at room temperature and incubated with primary antibodies against TSG101, CD9, CD63, CD81, GM130, Calnexin and β-actin (Abcam, Cambridge, UK) overnight at 4°C. After vigorous washing in TBS-T, the membrane was incubated with horseradish peroxidase (HRP)-tagged secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) for 2 h. The labelled proteins were visualized on X-ray film (AgfaPhoto, Germany) using a developer and fixer (VIVID, Korea). All chemical reagents and protease inhibitor cocktails were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Total proteins from cells, tissues, and exosome samples were extracted using a RIPA kit (Beyotime Biotechnology, Jiangsu, China). They were separated on polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-FOSL2 (ab124830; Abcam, Cambridge, UK) antibodies at 4°C overnight, and they were then incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse immunoglobulin G at room temperature for 1 h. To identify exosome markers, primary antibodies against TSG101 were purchased from Abcam (Cambridge, UK), and primary antibodies against Hsp 70 were obtained from Cell Signaling Technology (CST, Beverly, MA, USA). The secondary antibodies were F(ab)2 fragments of donkey anti-mouse immunoglobulin or donkey anti-rabbit immunoglobulin linked to horseradish peroxidase (Jackson ImmunoResearch Laboratories, USA). Proteins were visualized using Pierce ECL Western Blotting substrate and autoradiography. Blots were analyzed using Quantity One 4.6.