AGF and TIE receptors. AGF does not bind to tyrosine kinase with immunoglobulin and EGF-homology domain (Tie) receptors. (A) Schematic representation of the plasmid construction used to generate FLAG-AGF protein. CMV-p and SS indicate the cytomegalovirus promoter and a signal sequence, respectively. (B) After transfection of HEK293 cells with a mouse AGF cDNA with a 5′-terminal extension encoding a FLAG-tag (panel A), mouse AGF-FLAG fusion protein was detected in culture supernatants by Western blot analysis with an anti-FLAG antibody (left) and an antimouse AGF antibody (right), with or without 2-mercaptoethanol (2-ME). Lanes contain approximately 10 ng purified protein. Arrows indicate the monomer of FLAG-AGF protein. (C) Ties are not receptors for AGF. BIAcore binding assay of AGF (200 ng) to the Tie1 and Tie2 receptors. As a positive control, human Ang-2-6xHis-tagged protein (200 ng) specifically bound to the Tie2-Fc protein (460 ng), but not to the Tie1-Fc (460 ng). Error bar represents mean ± SD.