Beyoecl plus kit

After the total EV protein concentration was determined using the BCA protein assay, 30 µg of total protein was loaded on a 10–15% SDS-PAGE gel and subjected to electrophoresis. The separated proteins were transferred to PVDF membranes. Then, the membranes were incubated with primary anti-CD63 (1:1000, #ab134045, Abcam, Cambridge, UK), anti-CD81 (1:1000, #ab109201, Abcam, Cambridge, UK), and anti-TSG101 (1:1000, #ab125011, Abcam, Cambridge, UK). The membranes were subsequently incubated with secondary antibodies (1:5000, #111-035-045; Jackson ImmunoResearch, Madison, WI, USA). Protein expression was visualized using a BeyoECL Plus kit (#MA0186; Meilunbio, Dalian, Liaoning, China).

After total protein concentration was determined, AT-EVs—amounting to 30 μg protein—were loaded onto a 10%–15% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and electrophoresed. AT-EV proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes, incubated with primary anti-CD9 (1:1,000, #ab236630), anti-CD63 (1:1,000, #ab134045), anti-CD81 (1:1,000, #ab109201), anti-TSG101 (1:1,000, #ab125011), and anti-GM130 (1:1,000, #ab52649) antibodies (all obtained from Abcam, Cambridge, UK), followed by incubation with secondary antibodies (1:5,000, #111-035-045, Jackson ImmunoResearch, West Grove, PA, United States). Protein expression was evaluated using a BeyoECL Plus kit (#MA0186, Meilunbio, Dalian, Liaoning, China).