Sc 515727

Total protein was isolated using radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA). Equal amounts of protein were separated on 12% sodium dodecyl sulfate polyacrylamide gels and electro-transferred onto nitrocellulose membrane (Bio-Rad) at 100 V for 2.5 h at 4 °C. The membrane was blocked with 5% BSA for 1 h at RT and probed with primary antibodies against ACE2 (1:600, AF933, R & D Systems) and TMPRSS2 (1:1000, sc-515727, Santa Cruz). GAPDH (1:1000, 3683 S, Cell Signaling) was used as internal control. HRP-conjugated rabbit anti-goat (1:2000, 1721034, Bio-Rad) or goat anti-mouse (1: 2000, STAR117, Bio-Rad) were used as secondary antibodies. For immunoblotting of exosomal proteins, rabbit polyclonal CD63 antibody (1:1000, Abcam) and goat-anti-rabbit (1:2000, Cell Signaling) were used. Target proteins were visualized with an enhanced chemiluminescence detection system (GE Healthcare, NJ) using ChemiDocTM MP imaging system and associated software (Bio-Rad, Hercules, CA).

Cells were lysed on ice for 30 min in lysis buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, 1% Triton X-100) supplemented with protease inhibitor cocktail mini EDTA-free and PhoSTOP (Roche, Mannheim, Germany) and centrifuged at 16,000 × g at 4°C for 10 min to remove cell debris. Lysate concentrations were determined with Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher) using a Versa Max microplate reader (Molecular Devices, San Jose, CA). Protein samples (60 μg) were resolved on a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAG), and transferred onto 0.2-μm polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA), using standard procedures. The PVDF membranes were immunoblotted with a mouse monoclonal anti-actin antibody (MAB1501; Sigma) (diluted 1:20,000) and a mouse anti-TMPRSS2 antibody (sc-515727; Santa Cruz Biotechnology) (diluted 1:500). The blots were processed for chemiluminescence using a WesternBright Sirius kit (catalog no. K-12043-D20; Advansta, San Jose, CA) and imaged with the ChemiDoc system (Bio-Rad).