VEGFR2 Mediates Endothelial Permeability by OxPAPC Figure 6. Effect of VEGFR2 depletion on phosphorylation of MLC and VE-cadherin induced by high OxPAPC concentration. HPAECs were transfected with VEGFR2-specific siRNA. Control cells were treated with non-specific RNA. After 72 hrs of transfection, cells were stimulated with OxPAPC (75 mg/ml) or vehicle. A – Time-dependent phosphorylation of MLC and VE-cadherin in OxPAPC-stimulated EC was detected by western blot with diphospho-MLC and phospho-Y731-VE-cadherin specific antibodies. Equal protein loading was confirmed by probing of membranes with b- tubulin antibodies. B - Quantitative analysis of MLC and VE-cadherin phosphorylation. All experiments were repeated three times. Values are mean 6 SD, * p,0.05 vs control. C – Phosphorylation of MLC and VE-cadherin in HAEC treated with nonspecific and VEGFR2-specific siRNA and stimulated with OxPAPC (50 mg/ml, 30 min) was detected by western blot with diphospho-MLC and phospho-Y731-VE-cadherin specific antibodies. Probing with antibody to GAPDH was used as protein loading control. doi:10.1371/journal.pone.0030957.g006 PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e30957