Fig. 1

RON antibodies panel screened by ELISA, western blotting and cell staining. a 18 hybridoma clones chosen from 2112 hybridomas were screened against the RON1 and RON3 protein antigens in ELISA. GST was used as a negative control antigen. Absorbance was measured at 650 nm. b Three constructs were constructed as RON1, RON2 and RON3 antigens used for immunization in mice. The RON proteins were fused to GST tag at the N terminus and expressed recombinantly in E. coli cells. c Western blot analysis of two clones 3G4 and 6E6 on 293T cell lysate transfected with full length RON plasmid ran on 4–12% gradient gel. Lanes 1 and 3 (+) indicates cells transfected with full-length RON plasmid and lanes 2 and 4: (−) indicates untransfected cells. 3G4 and 6E6 bind to an epitope on alpha chain of RON, represented by pro-RON (180 kDa) and alpha-RON bands (35 kDa). d Immunofluorescence staining of H1299 cells transfected with full-length RON plasmid with a c-Myc tag using supernatants from hybridoma clones. Cells were stained with DAPI (blue) for nuclei visualisation and antibodies (green) using anti-mouse Ig secondary antibody conjugated with Alexa Fluor 488. Eight hybridoma clones were chosen for further development. Anti-c-Myc antibody 9E10 (Santa Cruz Biotechnology) was used as a positive control to visualize RON-transfected staining