2.5. Paxillin Staining

Non-transfected EC cells grown on fibronectin coated coverslips were fixed with cold 4% PFA for 15 min at RT and washed 3× 5 min with PBS. Unspecific binding was blocked with 10% FBS/PBS for 1h at RT. Anti-paxillin antibody (BD Biosciences, San Jose, CA, USA, #610619) was diluted in 1% FBS/PBS and incubated overnight at 4 °C. Cells were washed 3× 5 min with PBS and the incubated with donkey anti-mouse AlexaFluor 594 (Invitrogen, Waltham, MA, USA, www.Thermofisher.com) secondary antibody for 1 h at 4 °C, followed by 3× 5 min washes with PBS and PBS with 1 μg mL⁻¹ Hoechst 33342 for 15 min, followed by a quick wash and mounting on an objective glass. Confocal images of EC that had spread were taken and analyzed by ImageJ for fluorescence intensity in the peripheral area vs. the perinuclear area. The boundary between the perinuclear and peripheral was set at half the distance from the nucleus to the edge of the cell and the fluorescence intensity in each region determined. Ratios were calculated for each cell (10 wild type and 8 KO).