Properties of tPA-GFP expressed in EA.hy926 cells. Intracellular localization of intrinsic tPA (A) and forcibly expressed tPA-GFP (B) in confluent EA.hy926 cells analyzed by immunofluorescence staining and represented by both epifluorescence (epi-F) and total internal reflection-fluorescence (TIR-F) images. (A) Intrinsically expressed tPA molecules in EA.hy926 cells detected by anti-tPA antibody together with Alexa Fluor 488–labeled anti-IgG. (B) tPA-GFP (top panels) and tPA-related proteins detected by anti-tPA antibody together with TRITC-labeled anti-IgG (middle panels). The bottom panels are the merged images of the top and middle panels. The bars represent 10 μm. (C) Western blotting of 24-hour culture media from untransfected cells or transfected with tPA-GFP expression vector. Protein bands were detected by either anti-tPA or anti-GFP antibody. (D) The same samples in panel C were also analyzed by plasminogen-rich fibrin autography. M indicates molecular weight markers for 201, 120, 100, and 56 kD; R, a mixture of recombinant tPA and recombinant PAI-1; ◀, tPA-PAI-1 complexes; and ◁, free tPA. Extra-high-molecular-weight bands in culture medium from tPA-GFP–expressing cells () were detected by both anti-tPA and anti-GFP antibodies, and developed lytic bands in fibrin autography.