Anti gm130

The protein extracts were separated using 12% SDS‐PAGE and transferred onto a PVDF membrane (Millipore). The membranes were blocked with 5% evaporated skimmed milk in TBS (50 mmol/L Tris‐HCl, pH 7.5, 150 mmol/L NaCl) containing 0.1% Tween‐20 for 2 hours and incubated overnight at 4°C with the appropriate primary Ab, followed by incubation with HRP‐coupled secondary Ab for 1 hour at room temperature. Furthermore, the protein bands were visualized on photographic film using ECL blotting detection reagents (P0018; Beyotime). The following primary antibodies were used for western blotting: anti‐CD63, anti‐GM130, and anti‐TSG101 (Proteintech, America).

To validate whether the fusion protein was successfully expressed in 293T cells and integrated into the exosome membranes, we performed western blot using anti-His antibody. Total protein of isolated exosomes or cells was extracted in RIPA Lysis Buffer (Solarbio, China) at 4 °C for 15 min. The protein concentration was determined using a BCA protein assay kit (Solarbio, China). About 30 µg of protein was separated on 10% SDS-PAGE gels, and then transferred to nitrocellulose membranes (Millipore). The membranes were blocked with 3% BSA, and then incubated with anti-His-tag (1:5000, Abmart, China, #M20001), and anti-β-actin (1:4000, Sigma, St Louis, MO, USA) at 4 °C overnight. Anti-CD63 (1:2000, Proteintech, Wuhan, Hubei, China, #25682-1-AP), anti-GM130 (1:2000, Proteintech, #11308-1-AP), anti-TSG101 (1:2000, Proteintech, #28283-1-AP) were also used to characterize the exosomes. anti-LPCAT1 (1:2000, Proteintech, #16112-1-AP), anti-EGFR (1:5000, Proteintech, #18986-1-AP). This procedure was followed by adding HRP-conjugated secondary antibody (1:10000, Cell Signaling Technology, Beverly, MA, USA) and ECL reagents (Solarbio, Beijing, China). The bands were visualized and recorded using the Tanon 5500 imaging system.