RGDS internalization in melanoma cells. A: Bt-RGDS internalization into live SK-MEL-110 was examined after 24 h incubation at 37°C. Cells were treated with biotin alone or with increasing concentrations of bt-RGDS, cytoplasmic extracts were immobilized onto nitrocellulose and biotin presence was detected by avidin-peroxidase kit. Densitometry-quantification of total internalization in three experiments and one representative experiment are reported. B: Total internalization measured in extracts obtained from cells treated with bt-RGDS (50 μg/ml), and nonspecific internalization in the presence of an excess of unlabeled RGDS (1 mg/ml). Densitometry of three separate experiments and one representative experiment are reported. C: Bt-RGDS internalization (500 μg/ml) at 24 h was confirmed by confocal microscopy (original magnification × 40). RGDS entered into live melanoma cells (green stain) with a prevalently cytoplasmic localization. Nuclei are shown as blue stain. This experiment was carried out three times in duplicate.