Endocytosis of biotin-prekallikrein by ECV304 cells.

<p>ECV304 cells were grown on cover slips, and the lysosomes/endosomes were labeled with 0.5 μM LT Red DND-99 in incubation buffer for 20 min at 37°C. They were treated with or without H-kininogen (100 nM, unlabelled) for 1 h and then treated with biotin-prekallikrein (100 nM) for 1 h at 37°C. The cells were incubated with FITC-conjugated streptavidin (green). Alternatively, after labeling with 0.5 μM LT Red DND-99 at 37°C, the cells were maintained at 4°C and then treated with or without H-kininogen (100 nM, unlabelled) for 1 h and with biotin-prekallikrein (100 nM) for 1 h at 4°C. Biotin-prekallikrein (green) endocytosis and intracellular localization are indicated by their colocalization with acidic vesicles previously labeled with LT Red DND-99 (red) and were analyzed by confocal fluorescence microscopy. Normal endocytosis by ECV304 cells at 37°C without H-kininogen (A–C): biotin-prekallikrein (A), lysosomes/endosomes labeled with LT Red DND-99 (B), merged images and diphasic contrast (C); with H-kininogen (D–F): biotin-prekallikrein (D), lysosomes/endosomes labeled with LT Red DND-99 (E), merged images and diphasic contrast (F). Normal endocytosis by ECV304 at 4°C without H-kininogen (G–I): biotin-prekallikrein (G), lysosomes/endosomes labeled with LT Red DND-99 (H), merged images and diphasic contrast (I); with H-kininogen (J–L): biotin-prekallikrein (J), lysosomes/endosomes labeled with LT Red DND-99 (K), merged images and diphasic contrast (L).