Secretory dynamics of tPA-S478A-GFP and tPA-GFP in PAI-1 siRNA–transfected cells. (A) Twenty-four–hour culture media from tPA-GFP–expressing (wild type) or tPA-S478A-GFP–expressing (S478A) cells analyzed by Western blotting using anti-tPA antibody. M indicates molecular weight markers for 201, 120, 100, and 56 kDa; ◀, tPA-PAI-1 complex. Additional high-molecular-weight bands of tPA-GFP-PAI-1 complexes (), which reacted with anti-tPA antibody, were detected only in culture media from tPA-GFP–expressing cells. (B) Epi-F and TIR-F images in tPA-GFP– and tPA-S478A-GFP–expressing cells. tPA-S478A-GFP appeared diffusely distributed over the membrane surface as shown in the TIR-F image. The bars represent 10 μm. (C) Cells expressing either tPA-GFP (wild type, 11 cells) or tPA-S478A-GFP (S478A, 13 cells) were exposed to Mes-buffered pH 5.5 solution for 1 minute, and the decrease in relative fluorescence intensity in a whole cell was measured by epi-F image (dF_cell) calculated. Decrease in fluorescence intensity was significantly greater in tPA-S478A-GFP cells than tPA-GFP cells. Data are shown as mean plus or minus SD. (D) T_F1/2 values (abscissa) in tPA-GFP (■, 77 granules in 10 cells), tPA-S478A-GFP (□, 54 granules in 7 cells), and tPA-S478A-GFP supplemented with 40 nM rPAI-1 (, 12 granules in 2 cells) shown as percentages of total occurrences (ordinate). Exogenous PAI-1 did not modify the slow disappearance, having long T_F1/2 in tPA-S478A-GFP–expressing cells. (E) Eighteen-hour culture media from siRNA (PAI-1 no. 1, no. 4, and control)–transfected cells analyzed by Western blotting using anti–PAI-1 antibody. R indicates mixture of recombinant tPA-PAI-1 complexes and free PAI-1. (F) T_F1/2 values (abscissa) of tPA-GFP in control siRNA–treated (□, 113 granules in 8 cells, 5 experiments), PAI-1 siRNA no. 1–treated (■, 141 granules in 10 cells, 5 experiments), and PAI-1 siRNA no. 4–treated (, 40 granules in 3 cells, 3 experiments) cells, shown as percentages of total occurrences (ordinate). (G) T_F1/2 values (abscissa) of tPA-GFP before (■, 66 granules) and after (□, 30 granules) addition of 40 nM rPAI-1 in PAI-1 siRNA no. 1 or no. 4 transfected cells (3 experiments) shown as percentages of total occurrences (ordinate). Exogenous PAI-1 increased the frequency of rapid disappearing granules having short T_F1/2.