FIGURE 4.

A, Schematic representation of uPA variants and their relevant properties. WT, wild type; tcuPA, two-chain uPA; K, kringle; PD, protease domain, ΔGFD-uPA, GFD-deficient uPA; ΔK-uPA, Kringle-deficient uPA; LMW-uPA, low molecular weight (LMW) uPA missing the GFD and Kringle domains; ATF-uPA, N-terminal fragment of uPA consisting of the GFD and the kringle. B, nuclear translocation of uPA in hMLVEC depends on its kringle domain. Recombinant WT-uPA and ΔK-uPA, isotopically labeled with Na¹²⁵I as described (37), were incubated with hMVLEC for 1 h. Cells were then washed to remove unbound radioactive proteins and fractionated, and membrane-, cytoplasm- and nucleus-associated uPAs were quantified (37). Results are presented as the amount (fmol) of each protein found in each cellular fraction (membrane-bound, cytoplasmic, and nuclear) obtained from 10⁶ cells. All experiments were performed in triplicate. *, p < 0.05. C, nuclear localization of exogenously added WT-uPA, ΔGFD-uPA, and ΔK-uPA in hLMVEC (left panel). Cells growing exponentially on 8-well chamber slides were incubated with recombinant WT-uPA or ΔGFD-uPA or ΔK-uPA (10 nm) for 1 h, washed, and fixed in MeOH. uPA was detected using immunofluorescence as described under “Experimental Procedures” and in Stepanova et al. (37) with anti-uPA rabbit polyclonal antibodies and Alexa 488-conjugated secondary anti-rabbit antibodies. Nuclei were counterstained with DAPI and pseudo-colored in red. Green denotes positive staining for uPA. Nuclear localization of anti-uPA antibody-positive staining was quantified using Zeiss LSM 5 Image software. The white arrow (merge panels) was used to denote the profile quantified by the LSM 5 Image software. A graphic representation of the quantitative analysis of this profile for each treatment is shown on the bottom of the collage. The right panel shows staining obtained when control rabbit Ig (R Ig) was used as a negative control. Scale bar, 20 μm.