Transport of scuPA variants to the nucleus depends on their binding to nucleolin. (A) Subcellular distribution of ¹²⁵I-WT-scuPA, ¹²⁵I-ΔGFD-scuPA, and ¹²⁵I-ΔK-scuPA in BJ cells. BJ cells were incubated with 10 nM ¹²⁵I-WT-scuPA, ¹²⁵I-ΔGFD-scuPA, or ¹²⁵I-ΔK-scuPA for 1 hour at 37°C and washed, and the subcellular fractions were isolated as in Figure 2. The radioactivity in each fraction was measured and normalized per 10⁶ cells to compare the absolute amounts of proteins incorporated into each fraction. The results of 1 experiment, representative of 3 so performed, are shown. (B) Nucleolin mediates nuclear translocation of scuPA in the absence of LRP and uPAR. MEF-2 cells were preincubated for 1 hour in medium alone (control) or media supplemented with either 8 μM human lactoferrin, or 2.5 μM recombinant FLAG-tagged C-terminal fragment of m-nucleolin (GAR), or 5 μM recombinant full-length m-nucleolin. ¹²⁵I-WT-scuPA (10 nM) was added in the continued presence of potential competitors. The incubation was continued for an additional hour, and the radioactivity in the subcellular fractions was determined as in Figure 2. Nuclear transport of ¹²⁵I-WT-scuPA in control cells was taken as 100%. Nuclear contents of ¹²⁵I-WT-scuPA in cells pretreated with these potential inhibitors were expressed as percentage of control. Experiments were performed in triplicates, and data are presented as mean plus or minus SE. *indicates a result that is statistically significantly different (P < .05) from control.