Figure 3

RGDS-survivin interaction. A: Bt-RGDS interaction with cell lysate: cytoplasmic extracts were immobilized onto nitrocellulose and incubated with bt-RGDS. Total binding in the presence of labeled RGDS and nonspecific binding in the presence of an excess of unlabeled RGDS are shown. Densitometry of three different experiments and one representative experiment are reported. B: RGDS interaction with intracellular proteins was assayed by co-precipitation. BSA (control), bt-RGES (specificity control) or bt-RGDS (1 mM) were incubated for 1 h at 4°C with streptavidin-coated dynabeads, then SK-MEL-110 lysate, pre-incubated for 4 h at 4°C with an excess of unlabeled RGDS, was added and incubated overnight at 4°C. Precipitated proteins were revealed by western blotting using various antibodies (anti pro-caspase-8, anti pro-caspase-9, anti pro-caspase-1, anti pro-caspase-3 and anti-survivin). Total lysate was used as positive control. This experiment was carried out three times. C: Purified recombinant proteins were spotted onto nitrocellulose (0.9 μg/spot). Membrane was incubated for 4 h at RT with bt-RGDS (1 mg/ml) (total binding) in the absence or in the presence of an excess of unlabeled RGDS (10 mg/ml) (nonspecific binding). Three independent experiments were performed.