Fig. 4

Cellular uptake and interaction of ES variants with nucleolin. a ES variant internalization by endothelial cells determined by Western blotting. HMECs were treated with the indicated proteins, washed to remove cell surface-bound proteins, and used for Western blotting with anti-ES or anti-PEG antibodies to analyze the amount of internalized proteins. Heat shock protein 90α (Hsp90α) was used as the loading control. b Internalization of ES variants into endothelial cells detected by immunofluorescence. DAPI, 4′,6-diamidino-2-phenylindole. c Interaction of ES variants with nucleolin-turbo green fluorescent protein (nucleolin-tGFP) in vitro. Nucleolin-tGFP proteins were expressed in endothelial cells, and immunoprecipitated with anti-tGFP antibody and Protein G beads. The purified nucleolin-tGFP beads were incubated with 0.5 μg indicated ES variant at 4 °C for 1 h. Then, the beads were washed and collected, and Western blotting was used to detect the amount of bead-bound proteins. RR, relative ratio