Ab2501

Isolated MV were lysed in M-PER™ Mammalian Protein Extraction Reagent (Item78505, ThermoFisher) with protease inhibitors (Item78439, ThermoFisher). Total protein content of the lysates was measured by bicinchonic acid assay. A portion (20 µg total protein) of each sample was reduced using NuPAGE™ Sample Reducing Agent (ThermoFisher), resolved by PAGE, transferred to nitrocellulose, and probed with 2–5 µg/mL of primary antibody as previously described [36 (link)]. Antibodies used were rabbit pAbs recognizing laminin (Item L9393, Millipore-Sigma), col IV (Item ab6586, Abcam), fibronectin (Item AB2033, Millipore-Sigma), or TSG-6 (RAH-1; [37 (link)]). A rat monoclonal antibody recognizing human HSPG2 (perlecan) (Item ab2501, Abcam) was also used. Primary antibodies bound to the blots were visualized with the appropriate horseradish peroxidase‐conjugated secondary antibodies (Jackson Immuno Research, West Grove, PA) (1 µg/mL), visualized by enhanced chemiluminescence (GE Healthcare Life Sciences, Pittsburgh, PA), scanned, and quantified using ImageJ as previously described [36 (link)].

Human von Willebrand factor (VWF) native protein (RP-43132) was obtained from Thermo-Fisher Scientific (Pasley, UK). Cross-linked collagen-related peptide CRP was obtained from CambCol Laboratories (Cambridge, UK). Decorin isolated from bovine cartilage (D8428), human recombinant endostatin (GF171), and perlecan HSPG (H4777) were purchased from Sigma-Aldrich (Rotterdam, The Netherlands). Human recombinant endorepellin (2364ER) was obtained from R&D Systems (Abingdon, UK). Collagen Horm (collagen-H) derived from equine tendon was obtained from Nycomed (London, UK). BSA and hirudin (94581-1EA) were obtained from Sigma-Aldrich (Poole, UK). DiOC₆ (6975) was obtained from AnaSpec (San Jose, CA, USA). Fluorescein isothiocyanate (FITC)-labeled PAC1 monoclonal antibody (mAb) against activated integrin αIIbβ3 came from BD Bioscience (Franklin Lakes, NJ, USA). AF647-labeled anti-human CD62P mAb was obtained from Biolegend (San Diego, CA, USA). The anti-human endorepellin/perlecan Ab (AF2364-SP) was obtained from R&D systems (Abingdon, UK). The antibody against human heparan sulfate proteoglycan 2 and perlecan (Ab2501) was obtained from Abcam (Cambridge, UK). Secondary antibodies AF488-labeled anti-rat IgG and AF647-Germanylabeled anti-goat IgG were from Life Technologies (Paisley, UK).