Mouse monoclonal anti cd9

The PC cell lines PANC-1, MIA PaCa-2, and BxPC-3 were purchased from the American Type Culture Collection (Manassas, VA). Bovine growth serum, RPMI 1640, and DMEM medium were purchased from HyClone (Logan, UT). GEM was purchased from the pharmacy at Houston Methodist Research Institute and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Life Technologies (Grand Island, NY). Puromycin was purchased from Invitrogen (Carlsbad, CA). Mouse monoclonal anti-CD9, rabbit polyclonal anti-CD63, goat polyclonal anti-Stomatin, and mouse monoclonal anti-GAPDH antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal anti-TSG101 and rabbit monoclonal anti-GM130 antibodies were purchased from Abcam (Cambridge, MA). Mouse monoclonal anti-EphA2 antibody was purchased from Millipore (Billerica, MA). GIPZ Lentiviral EphA2 shRNAs were purchased from GE Dharmacon (Lafayette, CO).

The procedure for the isolation of exosomes from the culture medium of ADSCs (passage 3) was performed using an Exosome Isolation Kit (Invitrogen, USA). The exosomes isolated from ADSCs were pooled for bicinchoninic acid (BCA) assay, nanoparticle tracking analysis (NTA), protein separation and characterization, and Western blotting analysis. For the BCA assay (Thermo Scientific, USA) and NTA (JEM2100, JOEL Inc., Peabody, MA), the isolated exosomes were pelleted, fixed in 2.5% glutaraldehyde in cacodylate buffer at 20°C for 1 h, and stained with 2% uranyl acetate after three washes with phosphate buffered saline. The proteins in DMEM supplemented with 10% serum were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The exosomes produced by ADSCs in DMEM were purified, and the proteins in different exosome fractions (1 μg, 2 μg, 10 μg, and 50 μg) were also separated by SDS-PAGE. The gel was stained with Coomassie blue (Bio-Rad, Hercules, CA, USA) for analysis. For Western blotting analysis of the culture medium, conditioned medium, and exosome fractions, the following primary antibodies were used: mouse monoclonal anti-CD9 (Santa Cruz Biotechnology, USA), rabbit polyclonal anti-CD81 (TSG101; Abcam, USA), flotillin-1 (Abcam), and HSP70 (Abcam) antibodies.