FIG. 4.

Dasatinib and pazopanib block VE-cadherin internalization in ANDV-infected HUVECs. Human endothelial cells were mock infected or infected with pathogenic ANDV at an MOI of 0.5. Three days postinfection, endothelial cells were pretreated with kinase inhibitors dasatinib (1 nM/liter) and pazopanib (100 nM/liter) or left untreated for 15 min prior to VEGF addition (100 ng/ml, 2 h). Cells were incubated with an antibody to the extracellular domain of VE-cadherin (BV9; Clonetics) at 4°C for 1 h (23, 26, 31). After antibody removal and washing, cells were incubated at 37°C in 5% CO₂ (31) (1 h) to permit intracellular trafficking of antibody-bound VE-cadherin (23). Cells were subsequently washed with a mild acid solution (2 mM PBS-glycine, pH 2.0; three times for 5 min each) in order to remove VE-cadherin antibody that was not internalized or were left untreated (23, 26, 31). Cells were paraformaldehyde fixed and Triton X-100 permeabilized prior to incubation with an FITC-tagged anti-mouse secondary antibody and examined on an Olympus IX51 microscope. Data are presented as a percentage of cells with internalized VE-cadherin in ANDV-infected ECs pretreated only with VEGF (control) versus mock-treated infected cells (n = 500; P < 0.001) (31). Data represent the results from two independent experiments.