Figure 1

Mouse mAb Zt/f2 is highly specific and sensitive to human RON: A) Zt/f2 recognizes an epitope on RON extracellular sequences. 3T3-RON cells (0.5 × 10⁶ cells per sample) were incubated at 4°C for 45 min with 1 μg/ml of Zt/f2, Zt/#1, Zt/g4, or control mouse IgG, respectively. Goat anti-mouse IgG coupled with FITC were used as the detecting antibody followed by flow cytometry analysis. B) Zt/f2 does not recognize RON homologues from different species. Cellular proteins (300 μg per sample) from cell lines known to express RON homologues were incubated with Zt/f2 (10 μg per sample) overnight followed by addition of Protein G-Sepharose. Immunoprecipitated proteins were separated in 8% SDS-PAGE under reduced conditions. Rabbit IgG cross-reacting with RON homologues (Santa Crutz Biotechnology) from different species (50 μg cellular protein/sample from cell lines: human HCT116, monkey COS-1, canine MDCK, rat IEC18, and mouse peritoneal macrophages) were used followed by ECL reactions. C) Zt/f2 only recognizes RON but not other RTKs. Cellular proteins (300 μg per sample) from cell lines known to express MET (HCC1937), EGFR (BxPC3), VEGFR (HT-29), IGFR (AsPC-1), and FGFR (MDA-MB-361) were incubated with Zt/f2 (1 μg per sample) followed by immunoprecipitation with Protein G-Sepharose. Western blot analysis was performed as in B using specific antibodies. D) &E) Zt/f2 does not compete with MSP or Zt/g4 for RON. 3T3-RON cells were incubated with 3 nM of FITC-labeled Zt/f2 alone or with increased amounts of MSP (D) at 0, 5, 10, 20, 40, 60 nM, or Zt/g4 (E) at 0, 10, 25, 50, 100, and 160 nM, respectively. The fluorescent intensity of each sample was plotted in individual figures.