Mouse monoclonal anti cd9

Cell extracts were prepared as previously described [12 (link)]. Cell extracts (30 μg) or EVs (3 µg) were mixed with sample buffer 5× (1M Tris-HCl pH 6.8, 5% (w/v) SDS, 6% (v/v) glycerol, 0.01% (w/v) Bromophenol blue) without DTT (non-reducing conditions, used for CD9 and CD63 antibodies) or with 125 mM DTT (other antibodies). Samples were electrophoresed on 12% acrylamide gel at 150 V for 1 h and transferred to PVDF membrane at 100 V for 1 h. As internal control, actin or calnexin were used. Rabbit polyclonal anti-H-Ras antibody, mouse monoclonal anti-Tsg101 antibody, goat polyclonal anti-calnexin antibody were from Santa Cruz Biotechnology, mouse monoclonal anti-CD9 and mouse monoclonal anti-CD63 were from Abcam (Cambridge, UK), mouse monoclonal anti β-actin was from Sigma-Aldrich. Sheep anti-goat (Sigma), donkey anti-rabbit and sheep anti-mouse HRP-linked secondary antibodies (Sigma Aldrich) were probed according to manufacturer’s instructions. Immunoblots were detected by chemiluminescence using ECL system.

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).