Anti human cd9

PL and EV samples were prepared with non-reducing Laemli loading buffer³⁰ and denatured at 70°C for ten minutes. Samples were loaded in a 10% to 12% gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and proteins were separated by electrophoresis. The transfer was performed onto nitrocellulose membrane (GE Healthcare, Pittsburgh, Pennsylvania, USA) with humid conditions. A five-minute incubation with 0.2% (w/v) Ponceau-S (Sigma-Aldrich) and 3% (v/v) acetic acid solution (Sigma-Aldrich) was performed for total protein visualization.
Membranes were incubated for one hour with blocking buffer with 10% dry skimmed milk (Central Lechera Asturiana, Granda, Spain). Primary antibody incubation was performed overnight with 1/2,000 dilutions of the following antibodies: anti-human CD9 (Thermo Fisher) and anti-human CD63 (Abcam, Cambridge, UK). Secondary antibody one-hour incubation was performed with 1/2,000 diluted horseradish peroxidase (HRP)-coupled anti-mouse immunogloblin G (IgG) (Thermo Fisher). Chemiluminescence detection with Clarity Western ECL Substrate (Bio-Rad, Hercules, California, USA), C-DiGit Blot scanner, and Image Studio-Digits Software 4.0 (LI-COR Biosciences, Lincoln, Nebraska, USA) were used for membrane exposure and image processing.