Ab59477

Protein samples were prepared with radio-immunoprecipitation assay (RIPA) buffer (Beyotime). The concentrations of protein samples were analyzed using the BCA method. Protein samples (35 μg/lane) were loaded onto 10% separating gel and blotted onto a PVDF membrane (Millipore, Billerica, MA, USA). After blocking with 5% skimmed milk for 1 h at room temperature, the membrane was incubated with the primary antibodies overnight at 4 °C, including anti-c-myc (ab32072, Abcam, Cambridge, MA, USA), anti-N-cadherin (ab280375, Abcam), anti-vimentin (ab92547, Abcam), anti-METTL3 (ab195352, Abcam), anti-SOX2 (ab92494, Abcam), anti-TSG101 (ab125011, Abcam), anti-CD63 (ab134045, Abcam), anti-CD81 (ab59477, Abcam), and anti-β-actin (ab8226, Abcam). Afterwards, the membrane was incubated with the secondary antibody (Abcam) for 1 h at room temperature. The protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Pierce, Waltham, MA, USA), and the intensities of protein bands were analyzed using the Image Lab analysis software (National Institutes of Health, Bethesda, MD, USA). Western blot assay was conducted three times.

Total cell lysates were prepared using lysis buffer (Biosource International) supplemented with Phosphatase Inhibitor Cocktail 2 (P5726, Sigma Aldrich), Phosphatase Inhibitor Cocktail 3 (P044, Sigma Aldrich), Protease Inhibitor Cocktail (Sigma-Aldrich) and PMSF serine protease inhibitor (Sigma-Aldrich), and 10 µg of proteins were separated by SDS-PAGE using Bolt 4–12% Bis–Tris Plus gels (Invitrogen). Gels were transferred onto nitrocellulose membranes using Power Blotter Select Transfer Stacks and the Power Blotter System (Invitrogen). Membranes were blocked with I-Block reagent (ThermoFisher) and incubated with the following primary antibodies overnight at 4 °C: mouse monoclonal anti-calnexin (sc-23954, Santa Cruz); mouse monoclonal anti-CD63 (ab213090, abcam); mouse monoclonal anti-CD81 (ab59477, abcam); mouse monoclonal anti-GAPDH (GTX627408); mouse monoclonal anti-Cytochrome C (SC-13156); mouse monoclonal anti-HSP70/HSC70 (W27, SC-24). Membranes were then incubated with the appropriate peroxidase-conjugated secondary antibody, Goat anti-Mouse (G-21040). Images were acquired using Westar Hypernova ECL substrate (Cyanagen) and iBright Western Blot Imaging Systems (Invitrogen).