Cd81 bs 6934r

Protein extraction was performed in 20–40 μL, of lysis buffer (4% SDS, 100 mM Tris-HCl and 100 mM DTT); followed incubation at 95 °C for 5 min, the lysates were centrifuge at 17,000× g for 20 min at 4 °C. Protein quantification was performed with the Bradford assay (Merck KGaA, Darmstadt, Germany). Protein samples of 25–30 μg were analyzed by immunoblotting on 10% SDS-PAGE as previously described [48 (link)]. The primary antibodies were—CD81 (bs-6934R, Bioss Antibodies, Woburn, MA, USA), diluted 1:500; TSG-101 (sc-7964, Santa Cruz, CA, USA), diluted 1:400; Calnexin (sc-23954, Santa Cruz, CA, USA), diluted 1:400. Membranes were visualized with a Chemi Doc XRS system (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK) and images processed with ImageLab (BioRad, Hercules, CA, USA).

RIPA Buffer (20–40 μL) was added to a pellet for each species to extract proteins, incubating the solution for 15 min in ice. The cell lysates were centrifuged at 13,000 rpm for 5 min and the supernatant containing the proteins was recovered, according to Botta et al. 2019 [99 (link)]. Proteins were quantified at Qubit^® 3.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and 25–30 μg protein samples were analyzed by immunoblotting on 10% SDS-PAGE as previously described [100 (link)]. CD81 (bs-6934R, Bioss Antibodies, Woburn, MA, USA) diluted 1:500 and TSG-101 (sc-7964, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:400 were used as primary antibodies. A HRP-conjugated IgG was used as secondary antibody (Vector Labs, San Francisco, CA, USA) diluted 1:10000. The signal was detected using the enhanced chemiluminescence method following the manufacturer’s instructions (Amersham) using Chemi Doc XRS system (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK) and images processed with ImageLab (BioRad Laboratories Ltd., Hemel Hempstead, UK).