Anti cd63

Western blot analyses of EVs were performed as our previously described [20 (link)]. Following 8% SDS-polyacrylamide gel electrophoresis, the proteins were transferred to a polyvinylidene difluoride membrane and blocked with 2% BSA in TBS-T buffer for 1 h at room temperature. The membrane was incubated with anti-CD9 (1:1000, ABclone, Tokyo, Japan, #A19027) or anti-CD63 (1:1000, ABclone, #A5271) antibodies overnight at 4 °C. After washing off the free antibodies, the membrane was then incubated with HRP-conjugated anti-rabbit IgG antibody (1:5000, Sigma-Aldrich, St. Louis, MO, USA #A6154) for 1 h at room temperature. Signals were detected with ImmunoStar LD Reagents (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) using Amersham Imager 680 QC (Cytiva, Logan, UT, USA).

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitor. After quantification by BCA kit (Beyotime Biotechnology), the denatured protein was separated on SDS‐PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The immunoblots were blocked with 5% fat‐free milk and incubated at 4°C overnight with primary antibodies anti‐CD63 (1:1200, Abclone), anti‐PTEN (1:1000, Cell Signaling Technology), anti‐β‐actin (1:1000, Wuhan Boster Biological Technology, China), anti‐Akt and anti‐p‐Akt (Ser473) (1:1000, Cell Signaling Technology). After incubation with the secondary antibody, the bands were detected by enhanced chemiluminescence kit on in the Gel‐Pro Analyzer software.