Exoab hsp70a 1

MSCs were plated in six-well plates at a density of 2 × 10⁵ cells/well for 24 h and treated with exosomes at a concentration of 200 µg for 48 h. After removing the medium, cells were lysed in a buffer (P0013 and 1 mM phenylmethylsulfonyl fluoride (PMSF), Beyotime Institute of Biotechnology, Shanghai, China) for 30 min at 4°C. Exosome sample preparation was similar as before. The supernatant was harvested by centrifuging at 12,000 g for 5 min at 4°C. A bicinchoninic acid (BCA) kit was used to measure protein concentrations. The proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin (BSA) for 1 h, detected for anti-HSP70 (EXOAB-Hsp70A-1, System Biosciences, USA), anti-CD63 (EXOAB-CD63A-1, System Biosciences, USA), anti-TSG101 (28283-1-AP, Proteintech, USA), anti-IGFBP2 (11065-3-AP, Proteintech, USA), anti-IGF-2 (ab177467, Abcam, UK), anti-IGF-1R (9750T, Cell Signaling Technology, USA), and anti-p-IGF-1R (Tyr1135, 3918T, Cell Signaling Technology, USA) at 4°C overnight, and then incubated with anti-rabbit IgG conjugated with horseradish peroxidase (HRP) (SA00001-2, Proteintech, USA) for 1 h at room temperature. The Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) was used to scan and analyze the images.

For protein extraction, EV-pellets (obtained after ultracentrifugation of a pool of four plasma samples, total 6 ml) were resuspended in 100 μl lysis buffer containing 300 mM NaCl, 50 mM Tris pH 7.4, 0.5% NP-40 and anti-proteases cocktail (Roche, Indianapolis, IN, USA). Protein concentrations were determined by the Bradford assay (Bio-Rad, Hercules, CA, USA), and 15 μg total protein was submitted to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Ponceau staining was performed before blocking with 5% milk. Primary commercially available antibodies used were CD63 1:1,000 (CBL553, Millipore, Billerica, MA, United States), RAB27B 1:250 (HPA019849, Sigma-Aldrich), Flotillin 1:500 (ab41927, Abcam, Cambridge, MA, USA), HSP70 1:1,000 (EXOAB-Hsp70A-1, System Biosciences, Mountain View, CA, USA), TSG101 1:500 (ab4A10, Abcam, Cambridge, MA, USA) and RAB7A 1:1,000 (ab50533, Abcam, Cambridge, MA, USA).

EVs were concentrated from fraction 2 using ultracentrifugation at 110K g for 90 min, supernatants were aspirated, and EV pellets were resuspended in radioimmunoprecipitation assay (RIPA) lysis buffer (89901; Thermo Fisher Scientific) with protease inhibitor cocktail (78415; Thermo Fisher Scientific). Protein quantification was performed using the Pierce™ BCA Protein Assay (23225 and 23227; Thermo Fisher Scientific). Mini-Protean^®TGC™ wells were loaded with 5 μg of EV protein in Laemmli Sample Buffer (1610747; Bio Rad) or 10 μL of the Precision Plus Protein Kaleidoscope prestained standards (1610375; BioRad).
Gel electrophoresis was run, samples and standards were transferred to a nitrocellulose membrane, and the membrane was labeled for heat shock protein 70 (HSP70) or tumor susceptibility gene 101 (TSG101) (EXOAB-Hsp70A-1 and EXOAB-TSG101–1, System Biosciences). Appropriate secondary antibody was applied and stained using SuperSignal™ West Pico Plus Chemiluminescent Substrate (34580; Thermo Fisher Scientific). Images were taken with ChemiDoc™ XRS+ with Image Lab™ Software (BioRad).

Cells were lysed in TNE buffer (50 mM Tris–HCl (pH 7.4). 100 mM NaCl. 0.1 mM EDTA) and total cell lysates were separated by SDS-PAGE, transferred to PVDF membranes, immunoblotted with antibodies and visualized using an enhanced chemiluminescence detection system (Bio-Rad, Hercules, CA, USA). The antibodies used for immunoblotting were an anti-CD9 (EXOAB-CD9A-1), an anti-CD63 (EXOAB-CD63A-1), an anti-CD81 (EXOAB-CD81A-1), and an anti-HSP70 (EXOAB-HSP70A-1) from System Biosciences (Palo Alto, CA, USA).