Analysis of the transmigration of THP-1 cells through a monolayer of mock, ephrinB2, or ΔephrinB2-transfected PAECs. (A) Basal penetration through mock-transfected PAECs was 5-fold increased after MCP-1 stimulation and significantly inhibited by PAECs overexpressing ΔephrinB2 (**P < .01 vs MCP-1 stimulated mock-transfected PAECs; n = 8). (B) The number of MCP-1 stimulated THP-1 cells that were lodged in the PAEC monolayer was significantly increased if ΔephrinB2 was overexpressed by the PAECs (*P < .05 vs mock-transfected PAEC). (C) The MCP-1–induced transmigration of human monocytes through a HUVEC monolayer was inhibited by treatment with 4 μg/mL soluble monomeric ephrinB2 (**P < .01 vs control, n = 3). (D) An EphB4 receptor body–based immunofluorescence technique was applied to visualize the accumulation of ephrinB2 (D; arrowheads, red fluorescence) in ephrinB2-overexpressing PAECs after contact with THP-1 cells (D; * green fluorescence; scale bar represents 10 μm) transmigrating through a pore (D; arrow) in the porous membrane.