DEP-1 decreases uPAR expression. (A) Immunocytofluorimetric detection of cell surface uPAR in nonpermeabilized HUVECs overexpressing DEP-1 (gray line) compared with mock-transfected HUVECs (black line). DEP-1-positive cells were detected by monoclonal mouse anti-DEP-1 antibody. Filled gray bar represents IgG. Data are mean ± SEM. (B) Western blot for uPAR from EC lysates. HUVECs were seeded sparsely (5000 cells/cm²) and were allowed to grow for 24 hours in complete growth medium. Consequently, cells were transfected with DEP-1 siRNA, which resulted in an efficient and sustained reduction of DEP-1 expression compared with transfection with scrambled (scr)RNA (data not shown). Cells were then incubated in complete growth medium until all samples had reached confluence. All samples were lysed and obtained as described in “Western blotting.” Knockdown of DEP-1 yielded a 3.03- ± 0.46-fold increase in uPAR protein expression in confluent ECs. (C) HEK 293 or (D) HUVECs were transfected for reporter gene analysis (ie, Elk-1 activity, a downstream target of ERK1/2) with DEP-1 and/or ERK1 overexpressing plasmids (indicated as amounts of DNA in nanograms per milliliter). Cells were harvested 24 hours after transfection and analyzed for luciferase activity. pSRα was taken as empty plasmid (control). Data are mean ± SEM. *P < .05.