To confirm the success of EX isolation, ~ 30–40 μg of exosomal protein was loaded onto a polyacrylamide gel (5% stacking and 10% resolving gel). After running the gel at 90 V for 20 min, followed by 120 V for 60 min in running buffer, the proteins were transferred to a polyvinyl fluoride membrane (0.45 μm; Millipore) and blocked with 5% skim milk (BD, Lincoln, NE, USA) for 2 h. The membranes were incubated overnight with primary antibodies (Abs) against CD63, CD9, C81 (rabbit pAb; SBI, Palo Alto, CA, USA), pregnancy-specific globin (PSG1), nestin (NES), progesterone receptor [PGR] (rabbit pAb; Absin, Shanghai, China), L1 cell adhesion molecule [L1CAM] (rabbit pAb; Abcam, Cambridge, UK), and placental alkaline phosphatase [PLAP](mouse mAb; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4 °C. The Ab dilution ranged from 500- to 1000-fold times, as appropriate. The next day, the membranes were washed and incubated with secondary antibodies (goat anti-rabbit-HRP; Beyotime, Shanghai, China). The blotted proteins were visualized using an enhanced chemiluminescence kit (Pierce Biotechnology, Rockford, IL, USA), and the signal was detected using Saga (Sage Sciences, Lincoln, NE, USA).

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