Figure 5

uPAR^-/- ECs demonstrated changes in focal adhesion proteins FAK(P-Tyr925), Pax(P-Tyr118), integrins, and signal transduction: (A) WT cells adherent on Vn for 4 hr were stained for FAK(P-Tyr925) (green) and the nucleus (blue) and were imaged using an 100× objective. FAK(P-Tyr925) was found to be localized on focal adhesions (arrow) in WT cells. (B) uPAR^-/- ECs stained for FAK(P-Tyr925) demonstrated that FAK(P-Tyr925) was present not only along the cellular membrane (arrow) but also centrally in the cytoplasm (arrow). (C) Similar to (A), WT cells adherent on Vn were stained for Pax(P-Tyr118) (green) and DNA (blue). Images (100× objective) demonstrated that Pax(P-Tyr118) is present mainly along the focal adhesions and lamellipodia (arrow) in WT cells. Robust lamellipodia formation was observed. (D) Pax(P-Tyr118) (green) localization in uPAR^-/- ECs adherent on Vn occurred along the focal adhesions (arrow) of the circularly shaped cell as well as centrally within the cytoplasm (arrow). Lack of lamelllipodia formation was observed in uPAR^-/- ECs (B, D). (E) Immunoblot analyses of focal adhesion proteins and STAT1. Quiescent WT and uPAR^-/- ECs were seeded on Vn-coated 6-well plates and allowed to adhere for 4 hr. The medium was aspirated and the cell lysates were utilized for analyses. Levels of integrins β1, β3, and FAK(P-Tyr925) were determined by IP of 100 μg of cell lysates. Levels of integrin β1 and FAK(P-Tyr925) were enhanced in uPAR^-/- ECs compared to WT cells, while levels of integrin β3, total FAK, and paxillin were similar between WT and uPAR^-/- ECs. uPAR^-/- ECs showed decreased levels of STAT1 compared to WT cells. Tubulin served as a loading control. (F) The graph shows the levels of FAK(P-Tyr925/FAK), STAT1/tubulin, and Integrin β1/tubulin between WT and uPAR^-/- ECs. The values obtained represent the mean ± SEM of three independent assays. Significance levels (*) indicates p value of < 0.05 between WT and uPAR^-/- cells.