Nucleolin was identified as an endostatin-binding protein. (A) Eluted fraction of Ni-NTA or ES-Ni-NTA affinity chromatography with 500 mM sodium chloride was applied to SDS-PAGE (Coomassie blue staining) and IB. (B) Indicated concentrations of ES, NL, heparin, and sodium sulfate were incubated for 2 hours. ES-NL complex was precipitated by anti-NL antibody, and was applied to IB using anti-ES antibody. (C) Kinetic binding sensorgrams depict the real-time interaction of ES with NL at indicated concentrations by real-time surface plasmon resonance (SPR), and Kd for the interaction of ES with NL is derived to be 2.32 × 10−8 M from these curves. (D) Schematic represents the ES deletion mutants, which were designed to map the NL-binding site on ES. (E) The plasmids, encoding GST-fusioned deletion mutants of ES, were transfected into HMECs. After 48 hours, GST-ES deletion mutants were pulled down from cell lysate by glutathione-Sepharose beads, and applied to IB using anti-ES. (F) NL, which was co-pulled down, was detected by IB using anti-NL.
