Mouse monoclonal anti cd81

Western blotting was performed as described previously with modifications [32 (link)]. Cells or EVs were lysed in 1x RIPA buffer with protease inhibitors. The lysate was centrifuged and the supernatants collected. Mouse monoclonal anti-beta-actin (Sigma), mouse monoclonal anti-C3b (Thermo scientific), mouse monoclonal anti-properdin (Abcam), rabbit monoclonal anti-HSP70 (Abcam), rabbit polyclonal anti-CD59 (Abcam), rabbit polyclonal anti-CD55 (Santa Cruz Biotechnology), mouse monoclonal anti-CD63 (Santa Cruz Biotechnology), mouse monoclonal anti-CD81 (Santa Cruz Biotechnology), Rabbit polyclonal anti-CD46 (Santa Cruz Biotechnology), Rabbit polyclonal anti-Calnexin (Bioss Antibodies Inc.), and rabbit polyclonal anti-CD55 (Santa Cruz Biotechnology) were used.

For Western blotting analyses, cells were recovered, washed twice with PBS, and centrifuged again. Approximately 3 × 10⁶ cells were lysed at 4 °C in RIPA (Radio-Immune Precipitation Assay) buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% (v/v) NP-40, 0.1% (w/v) SDS (sodium dodecylsulfate), 0.5% (w/v) sodium deoxycholate) in the presence of a protease inhibitor mixture. Insoluble material was removed by centrifugation at 13,000× g for 10 min at 4 °C.
Aliquots of cell lysates (10–30 μg) or EVs (5 μg) were mixed with sample buffer 5× (1 M Tris-HCl pH 6.8, 5% (w/v) SDS, 6% (v/v) glycerol, 0.01% (w/v) Bromophenol blue) without DTT (dithiothreitol) (non-reducing conditions, used for CD9 detection according to the manufacturer’s instructions) or with 125 mM DTT (used for the other antibodies). Samples were boiled for 5 min, electrophoresed on 12% acrylamide gel and electrotransferred to PVDF (polyvinylidene fluoride) membrane. After 30 min incubation at room temperature with blocking buffer (5% BSA in TBS-Tween), membranes were incubated overnight with the following primary antibodies: goat polyclonal anti-Alix (Santa Cruz, USA), mouse monoclonal anti-CD9 (Abcam, Cambridge, UK), mouse monoclonal anti-CD81 (Santa Cruz, USA), goat polyclonal anti-calnexin (Sigma-Aldrich, St Louis, MO, USA) and anti β-actin (Sigma-Aldrich).