Western Blot Analysis of Endothelial Markers

For the determination of protein expression, cells were lysed in RIPA buffer supplemented with a cocktail of protease inhibitors (cOmplete™, Mini, EDTA-free, Roche, Basel, Switzerland). Western Blot analysis was performed, as previously described [27 (link)]. Briefly, 20 µg of proteins was separated on SDS-PAGE (Bolt™ 4–12% Bis-Tris mini protein gel, Thermo Fisher Scientific) and electrophoretically transferred to PVDF membranes (Immobilon-P membrane, Merck). Membranes were incubated for 1 h at RT in Tris-buffered saline solution (TBS; 50 mM Tris-HCl pH 7.5, 150 mM NaCl) containing 5% non-fat dried milk, then incubated overnight at 4 °C in TBST (TBS + 0.5% Tween) supplemented with 5% BSA and anti-ICAM-1, anti-VCAM-1, or anti-tissue factor (TF) purified rabbit polyclonal antibodies or anti-CD31/PECAM mouse monoclonal antibody (1:2000, Cell Signaling Technology, Beverly, MA, USA). Anti-vinculin (Merck) or anti-β-actin (Cell Signaling Technology) mouse monoclonal antibodies (1:2000) were employed as the internal standard. Immunoreactivity was visualized with SuperSignal™ West Pico PLUS Chemiluminescent HRP Substrate (Thermo Fisher Scientific). Western Blot images were captured with the iBright FL1500 Imaging System (Thermo Fisher Scientific) and analyzed with the iBright Analysis Software.