Overexpression of p120 in MVECs inhibits PMN transmigration, and real-time imaging of VE-cad during PMN transmigration in MVECs. (A,B) MVEC monolayers were infected with different doses of AdV-p120GFP or AdV-GFP, and stimulated for 4 hours with TNF-α before the PMNs were perfused. Hec-1—Alexa 568 mAb was used to detect surface VE-cad by immunofluorescence staining. Total accumulation of PMNs was similar on p120GFP- or GFP-transduced monolayers (A). Overexpression of p120GFP (high dose) strongly inhibited TEM (B; P < .001). Values represent the mean plus or minus SD from 3 different experiments. (C,D) TNF-α–activated MVEC monolayers were immunolabeled with VE-cad—Alexa 568 mAb and inserted into the flow chamber. PMNs were perfused for 5 minutes and coverslips were fixed, permeabilized, and stained with EEA-1 Ab (C) or PMNs were allowed to transmigrate for 10 minutes and analyzed by 2-channel live-time microscopy (D). As the neutrophil begins to transmigrate, VE-cad forms a gap at the junction (panel 00:00) that widens (panel 02:00) and reseals (panel 03:00; yellow arrows), without apparent internalization of vesicles of VE-cad or recycling to the membrane of constitutively internalized VE-cad vesicles (thin white arrows). Merge panels show colocalization of the gap and the PMNs during the TEM process. Bar represents 10 μm. The figure represents a typical sequence of events during PMN transmigration from 3 independent experiments using HUVECs and PMNs from multiple donors.