Nucleolin-mediated endostatin signal pathway. (A) ES, which bound to the cell surface of siRNA-transfected HMECs, was stained with saturating amounts of monoclonal antibodies recognizing ES, and was evaluated by flow cytometric analysis. Scramble siRNA-transfected HMECs, which were treated with FITC-labeled goat IgG or anti-ES antibody, serve as negative or positive control, respectively. (B) After siRNA transfection for 48 hours, the HMECs were incubated with ES for 2 hours. ES, which was internalized into nucleus by siRNA-transfected HMECs, was detected by IB with the anti-ES antibody. Actin blot serves as loading control. (C,D) HMECs, which were incubated with ES for indicated time course, were separated into cytosol and nuclear fractions. The amount of ES in cytosol (C) or nucleus (D) was detected by IB with the anti-ES antibody, respectively. Lamin B and tubulin β serve as fraction controls for nuclear and cytosol, respectively. (E,F) HMECs (E) or separated nuclei of HMECs (F), which were treated with indicated reagents, were applied to IP with the anti-NL antibody. The serine-phosphorylated NL was recognized by the antibody against phosphorylated serine residues. NL blot serves as inputting control. Densitometry analysis of IBs showed the relative phosphorylation level of NL (p-NL/NL).