Figure 5.

Effect of phosphorylation on the ability of pro-uPA to promote adhesion of differentiating U937 cells. (A) U937 cells were grown to 0.8 × 10⁶ cells/ml and then diluted 1:2 and treated with TGF-β/vitamin D₃ for 20 h. Then, they were incubated with the following effectors at the concentration of 1 nM: His-pro-uPA^wt, His-pro-uPA^138E, His-pro-uPA^138E/303E, Ser-uPA (nonphosphorylated pro-uPA), Pser-uPA (phosphorylated pro-uPA), and DFPPser-uPA (DFP-inactivated Pser-uPA). Control samples included a combination of 5 nM His-pro-uPA^138E/303E and 1 nM His-pro-uPA^wt (WT+138E/303E) and a preincubation of the cells with 10 μg/ml anti-uPAR polyclonal antibody for 1 h before the addition of 1 nM His-pro-uPA^wt (WT+399Ab). The number of adherent and non-adherent cells was counted 30 min later and reported as a percentage of the maximal adherence observed (37.5% of the total cell number, with 1 nM His-pro-uPA^wt, over a background of 12.6% due to the TGF-β/vitamin D₃ addition). The data represent the average of three experiments performed in duplicate with standard deviations indicated by error bars. (B) U937 cells were primed for the previous experiment and incubated with increasing concentrations of His-pro-uPA^wt (□) or His-pro-uPA^138E/303E (○). The basal adherence of TGF-β/vitamin D₃-treated cells was 11.7 (SD = 2.12). The number of adherent cells is reported as a percentage of the total cell number.