Subcellular distribution of cell-associated ¹²⁵I-WT-scuPA and ¹²⁵I-ΔGFD-scuPA. BJ cells were in-cubated with ¹²⁵I-WT-scuPA (A-D) or ¹²⁵I-ΔGFD-scuPA (E) in complete medium at 37°C at the indicated concentrations for 1 hour and then washed to remove unbound radioligand. Surface-bound uPA was eluted with glycine buffer, pH = 3.0 (surface). Subcellular fractions were then prepared as described in “Methods,” and the radioactivity in each fraction was counted. Radioactivity in the cytoplasmic extract and wash fractions were combined as a measure of total cytoplasmic content. Radioactivity in the nuclear extract and nuclear pellet were counted separately, but the values were combined as a measure of total nuclear content. (A) Distribution of nuclear and cytoplasmic cell markers. The cytoplasmic extract, the 2 wash fractions, and the nuclear extract were analyzed by SDS-PAGE under reducing conditions, electrotransferred to nitrocellulose membranes, and probed with antibodies to the indicated proteins. This figure indicates that the nuclear fraction does not contain detectable amounts of plasma membrane and cytoplasmic proteins or markers of organelles. (B) Subcellular distribution of ¹²⁵I-WT-scuPA. BJ and HeLa cells were incubated with ¹²⁵I-WT-scuPA, cell fractionation was performed as described in “Methods,” and the radioactivity in each fraction was measured. Total cell-associated radioactivity was designated as 100%. (C) ¹²⁵I-WT-scuPA does not degrade/becomes cleaved upon translocation to the nucleus. BJ cells were incubated with 20 nM ¹²⁵I-WT-scuPA for 1 hour. The subcellular fractions were isolated as above. The proteins were separated by SDS-PAGE and analyzed using autoradiography under nonreducing (NR) and reducing (1 mM DTT) (R) conditions. Input lane: ¹²⁵I-WT-scuPA. This figure shows that cytoplasmic and nuclear scuPA migrates as a single band that corresponds to intact protein, and no additional low-molecular-weight bands were evident. (D,E) Dose-dependent accumulation and subcellular distribution of¹²⁵I-WT-scuPA (D) and ¹²⁵I-ΔGFD-scuPA (E). The experiments were performed as in panel B. All experiments were performed in triplicate and repeated 2 to 3 times. Data from a representative experiment are presented as mean plus or minus SE.