Figure 9

Nucant pseudopeptides bind surface nucleolin at a higher affinity compared to binding to nucleophosmin. A. Recovery of surface nucleolin and associated proteins by using biotinylated Nucant. MDA-MB 231 cells were incubated in the absence (lanes Control) or presence of 6 μM biotinylated N3 (N3/Biot.) or 4 μM of N7 (N7/Biot.) for the recovery of surface nucleolin and associated proteins under similar experimental conditions as described in the case of HB-19/Biot. (Methods). Purified material was then analyzed by SDS-PAGE and stained with Brilliant Blue G-Colloidal Concentrate staining (panel Staining). Similar samples were also analyzed by immunoblotting using anti-nucleolin mAb D3 (panel anti-nucleolin) or anti-nucleophosmin mAb EP1848Y (panel anti-nucleophosmin)(Methods). B. Nucant binds nucleophosmin independently of its capacity to bind to surface nucleolin. The T-cell leukemia HuT 78 cells were incubated at 0, 1, 2, 4, and 8 μM of the biotinylated N7 for the recovery of surface nucleolin and associated proteins. Nucleolin and nucleophosmin were revealed by immunoblotting as above.