Binding and accumulation of plg-568 on tPA-GFP–expressing EA.hy926 cells. (A) After tPA-GFP–transfected cells were incubated with 480nM NL-plg plus 20nM plg-568 at 37°C for 30 minutes, localization of tPA-GFP and plg-568 was determined by total internal reflection fluorescence (TIRF) microscopy. Both fluorophores were excited at 488 nm, and the light emitted was collected through an emission splitter (W-View system; Hamamatsu Photonics) equipped with a 550-nm dichroic mirror and 2 emission filters, a 510/23-nm band-pass filter for GFP and a 600-nm long-pass filter for Alexa Fluor 568. Representative images are shown. Left panel shows epifluorescence image obtained only by GFP channel; others are TIRF images of tPA-GFP and/or plg-568. In addition to the high fluorescence intensity of tPA-GFP in possible Golgi complex, TIRF images demonstrated a number of clear spots, some of which colocalized with plg-567 (arrowheads). Bars represent 10 μm. (B) Expanded images of the area indicated by a white box in panel A. (C) Representative images captured by the CLSM system are shown. The plasma membrane was labeled with Cell Mask Deep Red plasma membrane stain in tPA-GFP–expressing cells, which were incubated with 480nM NL-plg plus 20nM plg-568 for 60 minutes. Top panels are the most basal focal images (the “bottom”), and bottom panels are optical images taken along the z axis on a 1.65 μm focal plane from the bottom. PM indicates plasma membrane stain. plg-568 (red) accumulated not only in the plasma membrane area (white) but also in the pericellular/matrix adhesive area in tPA-GFP–expressing cells. Bars represent 10 μm. (D) Time-dependent accumulation of plg-568 in each focal plane. The values of fluorescence intensity at every 10 minutes were normalized to that at the bottom at 60 minutes; relative fluorescence changes are referred to as F/F_{60 min}, bottom. The plots are indicated in each focal plane as mean ± SE of 7 independent experiments.