RGDS cell internalization and molecular interaction. (A) Biotinylated RGDS (500 μg/mL) penetration in live HUVECs was examined after 4 hours of incubation at 4°C and 37°C. Biotinylated RGDS internalized is shown as green stain (fluorescein-avidin staining), and nuclei are shown as red stain (PI staining, 5 μg/mL), by confocal microscopy (original magnification × 110). A short incubation (4 hours) was sufficient for RGDS to enter cells at 37°C incubation, whereas it did not penetrate at 4°C incubation. (B) Overlay assays were carried out with HUVEC membranes and cytoplasmic extracts immobilized onto nitrocellulose and incubated with biotinylated RGDS. Total binding was shown in the presence of labeled RGDS, whereas nonspecific binding was shown in the presence of an excess of unlabeled RGDS. This experiment was performed 3 times with similar results. A representative experiment is shown. (C) The binding was quantified by densitometry; the nonspecific binding was subtracted from the total binding, giving the values reported as specific binding. Data are expressed as mean ± SE of 3 experiments. (D) Overlay assays were carried out with biotinylated RGDS spotted onto immobilized proteins. Labeled RGDS specifically interacted with human recombinant active caspase 9 immobilized onto nitrocellulose (0.1 to 5 units/spot) and with human recombinant active caspase 8 (1 to 10 units/spot), after 4 hours of incubation at RT, whereas no interaction was observed with immobilized control proteins (BSA and fibronectin, 10 μg/spot). Total binding (ie, binding observed in the presence of labeled RGDS only) and nonspecific binding (ie, binding observed in the presence of labeled RGDS and an excess of unlabeled RGDS) are reported. This experiment was performed 2 times with similar results. A representative experiment is shown.