Fig. 1

Localization and identification of targeted antigen recognized by mAb 12C7. a Fixed SPCA-1 and A549 sphere cells were labeled with mAb 12C7 (green) and counterstained with the nuclear stain DAPI (blue). Fluorescent microscope images (× 1000) showed that targeted antigen is abundantly localized in the membrane, cytoplasm and nucleus. Scale bar, 10 μm. b Live SPCA-1 and A549 sphere cells were labeled with mAb 12C7 (green), after fixing and washing, counterstained with the nuclear stain DAPI (blue). Images (× 1000) further verified the cell membrane immunoreactivity. Scale bar, 10 μm. c Purification of targeted antigen recognized by mAb 12C7. Purified antigen proteins recognized by mAb 12C7 from cell lysate of SPCA-1 sphere cells were electrophorized (right panel) and then immunoblotted with mAb 12C7 (left panel). Results presented a band of 43–55 kDa protein. d The 43–55 kDa protein band was excised from SDS-PAGE and identified as ENO1 by mass spectrometry (right panel) and analysis in Mascot database (left panel). e Immunofluorescence co-localization of targeted antigen in SPCA-1 and A549 cells. Cells were co-incubated with mAb 12C7 (green) and commercial antibody to ENO1 (red), and counterstained with the nuclear stain DAPI (blue). Fluorescent microscope images (× 1000) showed the high coincidence between antigens labeled with mAb 12C7 (green) and with commercial antibody to ENO1 (red). Scale bar, 10 μm. f Identification of mAb 12C7-binding antigen by immunoprecipitation-western blot. Immunoprecipitation was performed using purified mAb 12C7 or commercial antibody to ENO1, followed by western blotting against ENO1. Results confirmed that a 43–55-kDa protein band of ENO1 antigen interacting with mAb 12C7