Fig. 1

a. Simulation structure of EN2. b. C-terminal of EN2 used as immunogen. c. Total proteins of 293 T transfected with or without EN2-RFP-expressing plasmid. Lane M was the protein marker, lane “EN2-RFP+” was 293 T cell transfected with EN2-RFP, as the arrow points out, the band was the EN2-RFP fusion protein. Lane “EN2-RFP-” was the total 293 T cell protein. d. Western blot analysis of extracts from 293 T cells with or without transfection of EN2-RFP by homemade EN2 monoclonal antibody. The lane on the left was 293 T cells with EN2-RFP fusion protein (+) while the lane on the right was 293 T cells without EN2-RFP fusion protein (−). The molecular weight of EN2-RFP fusion protein was 40 KDa while the molecular weight of endogenous EN2 was 33 KDa. e. WB of EN2 in LNCap, DU145 and PC3 cell lines with homemade EN2 monoclonal antibody. Total cell proteins were extracted and used. Only one band at 33 KDa appeared in extracts of all three PC cell lines. f. Immunofluorescence assay with homemade EN2 monoclonal antibody. From left to right: the image “ EN2-RFP “ was 293 T cells transfected with EN2-RFP, the image “anti-EN2-FITC” was 293 T cells stained with homemade EN2 monoclonal antibody, the image “isotype antibody” was 293 T cells stained with an control antibody, the image “merge” was merged image of “EN2-RFP “and “anti-EN2-FITC” or “isotype antibody”. Zoom in× 400. EN2-RFP fusion protein gave off red fluorescence, EN2 monoclonal antibody gave off green fluorescence since FITC was labeled at the second detection antibodies, the merged region in “merge” turned yellow in color. Full-length gels and blots are presented in Supplementary Figure 1,2 and 3. These experiments were repeated independently 3 times with similar results