Western blotting luminol reagent

Western blot analysis for placental alkaline phosphatase, Tumor Suspectibility Gene 101 (TSG101) and CD63, which are enriched in exosomes, was carried out. Total protein was extracted using the lysis buffer Pro‐Prep (iNtRON Biotechnology, Seongnam, Korea), and 20 μg of protein was separated using a 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis transferred onto a polyvinylidene fluoride (PVDF) membrane (GE Healthcare, Piscataway, NJ, USA). The membrane was then incubated with primary antibodies, namely anti‐placental alkaline phosphatase (1:2,000, ab95462; Abcam, Cambridge, UK), anti‐TSG101 (1:3,000, ab125011; Abcam) and anti‐CD63 (1:2,000, ab59479; Abcam), followed by incubation with a horseradish peroxidase‐conjugated anti‐mouse or anti‐rabbit secondary antibody (1:10,000; Novus Biologicals, Littleton, CO, USA). Proteins were detected using Western Blotting Luminol Reagent (Bio‐Rad, Hercules, CA, USA).

Cell protein samples (40 µg) were separated on an SDS-polyacrylamide gel and electrotransferred to polyvinylidene fluoride membranes. The membranes were then incubated overnight with primary antibodies at 4℃, washed with Tris-buffered saline with Tween 20 and further incubated with horse radish peroxidase-conjugated second antibodies at room temperature for 2 h. Antibody-antigen complexes were detected using Western blotting luminol reagent (Bio-Rad Laboratories, USA). Finally, reactive bands were quantified using the Quantity One software (ver. 4.6.2; Bio-Rad Laboratories). GADPH was used as the loading control.