Nbp2 42225

The collected EVs were lysed in Pierce immunoprecipitation lysis buffer containing 1× protein inhibitor (Roche) and 1 mM phenylmethylsulfonyl fluoride (Thermo Fisher Scientific), and the protein concentration was quantified using the bicinchoninic acid assay (BCA assay; Thermo Fisher Scientific). Protein lysates were resolved by SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (Invitrogen). The PVDF membrane was blocked with 5% nonfat dry milk in tris-buffered saline buffer for 1 hour at room temperature (RT) and then immunoblotted with 500-fold diluted primary antibodies overnight at 4°C. The primary antibodies used in this study included mouse anti-human MCSP (R&D Systems, MAB2585), MCAM (R&D Systems, MAB932), ErbB3 (R&D Systems, MAB3482), LNGFR (R&D Systems, MAB367; or Santa Cruz Biotechnology, sc-271708), calnexin (Abcam, ab112995), and CD63 (Novus, NBP2-42225). Proteins were analyzed under denaturing and reducing/nonreducing conditions according to the manufacturer’s instructions. After incubation, the PVDF membrane was washed with phosphate-buffered saline (PBS) containing 0.1% Tween 20 and then incubated with IRDye 800CW–conjugated goat anti-mouse (LI-COR, 926-32210, 10,000-fold dilution) for 1 hour at RT. After washing, protein bands were detected using the Odyssey LI-COR CLx Imaging System.