Figure 1
Figure 1. Characterization of p120 expression in vascular endothelium and p120GFP/VE-cad gap formation during leukocyte TEM at the endothelial cell junctions. Confluent HUVECs were transduced with GFP or p120GFP adenovirues as described in “Adenovirus production and cell infection.” (A) p120GFP colocalizes with endogenous p120 at cell junctions. Monolayers were transduced with p120GFP, or sham treated, fixed with 10% buffered formalin, permeabilized, and stained with anti-p120 mAb. Representative fields were examined by epifluorescence microscopy and show junctional distribution of p120GFP and colocalization with endogenous p120 at cell junctions. (B) VE-cad was immunoprecipitated from the HUVEC lysates, and the material was immunoblotted for p120 to show endogenous isoforms of p120/p100, or with an anti-GFP mAb to detect expression of p120GFP. (C) HUVECs were surface biotinylated, lysed, and subjected to immunoprecipitation with anti-p120 mAb, or VE-cad mAb. The association of p120GFP with VE-cad was detected with streptavidin-peroxidase. Vertical lines have been inserted to indicate a repositioned gel lane. (D) Transduced HUVECs were lysed directly and blotted with Hec-1 to detect total VE-cad. Normalized VE-cad values versus β-actin are shown by OD numbers. Data are representative of 3 separate studies. (E) Three-channel live-time microscopy of PMNs in the process of transmigration. Paired 2-color fluorescence of VE-cad Alexa 568–stained HUVECs (red channel), p120GFP low-dose infected HUVECs (green channel), and simultaneous DIC images are presented. At t = 0, PMN approaches the brightly stained cell junction. At t = 1:00, PMN starts to transmigrate and a de novo gap is detected. At t = 3:00, the gap is sealed. This gap in p120-catenin GFP colocalized with the gap formed by VE-cadherin (red) during transmigration, as demonstrated in the merge panel. Bar represents 10 μm. The figure represents a typical sequence of events during PMN TEM. Five independent experiments using HUVECs and PMNs from multiple donors were analyzed.

Characterization of p120 expression in vascular endothelium and p120GFP/VE-cad gap formation during leukocyte TEM at the endothelial cell junctions. Confluent HUVECs were transduced with GFP or p120GFP adenovirues as described in “Adenovirus production and cell infection.” (A) p120GFP colocalizes with endogenous p120 at cell junctions. Monolayers were transduced with p120GFP, or sham treated, fixed with 10% buffered formalin, permeabilized, and stained with anti-p120 mAb. Representative fields were examined by epifluorescence microscopy and show junctional distribution of p120GFP and colocalization with endogenous p120 at cell junctions. (B) VE-cad was immunoprecipitated from the HUVEC lysates, and the material was immunoblotted for p120 to show endogenous isoforms of p120/p100, or with an anti-GFP mAb to detect expression of p120GFP. (C) HUVECs were surface biotinylated, lysed, and subjected to immunoprecipitation with anti-p120 mAb, or VE-cad mAb. The association of p120GFP with VE-cad was detected with streptavidin-peroxidase. Vertical lines have been inserted to indicate a repositioned gel lane. (D) Transduced HUVECs were lysed directly and blotted with Hec-1 to detect total VE-cad. Normalized VE-cad values versus β-actin are shown by OD numbers. Data are representative of 3 separate studies. (E) Three-channel live-time microscopy of PMNs in the process of transmigration. Paired 2-color fluorescence of VE-cad Alexa 568–stained HUVECs (red channel), p120GFP low-dose infected HUVECs (green channel), and simultaneous DIC images are presented. At t = 0, PMN approaches the brightly stained cell junction. At t = 1:00, PMN starts to transmigrate and a de novo gap is detected. At t = 3:00, the gap is sealed. This gap in p120-catenin GFP colocalized with the gap formed by VE-cadherin (red) during transmigration, as demonstrated in the merge panel. Bar represents 10 μm. The figure represents a typical sequence of events during PMN TEM. Five independent experiments using HUVECs and PMNs from multiple donors were analyzed.

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