Anti cd63

Protein quantification was performed using the BCA Protein Assay Kit (P0011, Beyotime, Shanghai, China). Sample proteins were loaded onto a 10% SDS-PAGE gel, electrophoresed, and transferred to a PVDF membrane (0.45 µm; Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk, the membrane was probed with specific antibodies overnight at 4 ℃, including anti-CD63 (A5271, ABclonal, Wuhan, China) and anti-TSG101 (A1692, ABclonal, Wuhan, China). On the following day, the membranes were incubated with the appropriate secondary antibody (ZB-5301, ZSGB-BIO, Beijing, China) at room temperature for 1 h. Immunoblots were then visualized using the BeyoECL Plus Kit (P0018, Beyotime, Shanghai, China).

Proteins from exosomes and endothelial cells were collected and extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). The lysate was centrifuged at 12 000 rpm to obtain protein extracts. The protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime). Next, 15 μg of protein was loaded, separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Millipore, Mississauga, Canada). The membranes were incubated with the primary antibodies overnight at 4°C after being blocked with 5% nonfat milk for 1 h. Then, they were incubated with the appropriate secondary antibodies at room temperature for 1 h. The following primary antibodies were used: anti‐CD9, anti‐CD63, and anti‐TSG101 (1:1000; Abclonal, Woburn, MA, USA); anti‐SPRED1, anti‐Ras, and anti‐p/t‐Raf (1:1000; Abcam, Cambridge, UK); anti‐p/t‐MEK1/2 and anti‐p/t‐ERK1/2 (1:1000; Bioworld, Nanjing, China); and anti‐β‐actin (1:10000; Proteintech, Rosemont, IL, USA). The Tanon‐5200 Chemiluminescent Imaging System (Tanon Science & Technology, China) was used to detect the protein bands, after which the grayscale value was calculated using Image J software.

Exosomes were lysed with RIPA Lysis Buffer I (Sangon Biotech, Cat: C500005) to obtain the total protein. Then, the extracted proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The primary antibodies used here included: anti-CD9 (Abclonal, Cat: A1703), anti-CD63 (Abclonal, Cat: A5271), anti-TSG101 (Abclonal, Cat: A1692), and anti-HSPA5 (Abclonal, Cat:A11366).