Endostatin

Protein was extracted from total cell lysates by using RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) or from 48 h cell supernatant. Protein concentrations were measured with the Pierce BCA Protein Assay Kit (ThermoScientific). Twenty-five micrograms of protein was resolved by 1-DE under reducing conditions onto 10% SDS-PAGE gels and electrotransferred to nitrocellulose membranes. After blocking for non-specific binding with 5% of bovine serum albumin (BSA; MP Biomedical) or Blotto, the membranes were incubated with primary antibodies, including TF (4501-Sekisui Diagnostics), endostatin (ab109660-abcam), and VEGF (ab51745-abcam). Band detection was performed using a chemiluminiscent substrate dye (SuperSignal® West Dura Extended Duration Substrate, Thermo Scientific, Waltham, MA, USA) and a molecular imager ChemiDoc XRS System, Universal Hood II (BioRad, Hercules, CA, USA). Band quantification was performed with Image Lab 4.0 software (BioRad Laboratories, Hercules, CA, USA). Protein load was normalized with total protein staining, and normalization between different membranes was performed with a common pool in every gel, as previously described [24 (link)].

Specific ELISAs were used asper the manufacturers’ (all from R&D Systems unless otherwise stated) instructions to assess the plasma levels of TNF-α, granulocyte colony-stimulating factor (G-CSF), PDGF-BB, stromal derived factor-1 (SDF-1), total anti-oxidant capacity (Abcam), VEGF, endostatin, angiostatin (Abcam), thrombospondin-1, and thrombospondin-2.