Density-dependent uPAR expression is mediated by ERK/MAPK activity. (A) Western blots for phospho-(p)-ERK1/2 and pan-ERK1/2 from EC lysates. All samples were lysed in the same volume and prepared as described in “Western blotting” irrespective of cell density, and the same volume of samples was loaded. Consequently, the amount of loaded protein is higher in the 20 000 cells/cm2 sample than the 5000 cells/cm2 sample, reflected by the stronger signal in pan-ERK. Proteins were separated on a sodium dodecyl sulfate–10% polyacrylamide gel, and chemiluminescence of phosphorylated and pan-ERK1/2 was quantified with FluorTech HD2 from Alpha Innotech. (B) Immunocytofluorimetric detections of cell surface uPAR (nonpermeabilized cells; gray) and total (permeabilized cells; black) uPAR in HUVECs overexpressing either enhanced green fluorescence protein (EGFP) tagged ERK1 or EGFP alone. Transfected HUVECs were gated via EGFP fluorescence. Data are mean ± SEM. **P < .01.
