Fig. 2

Subcellular localization of exogenous and endogenous EN2 proteins in three PC cell lines. From top to bottom were LNCap, DU145, PC3 and LNCap cell lines. From left to right: the images “EN2-RFP” were LNCap, DU145, PC3 and LNCap cell lines transfected with EN2-RFP, the images “Anti-EN2-FITC” were LNCap, DU145, PC3 cell lines stained with homemade EN2 monoclonal antibody, the image “isotype antibody” was LNCap cell line stained with an isotype antibody as a negative control, the images “merge” were merged images of “ EN2-RFP “ and “Anti-EN2-FITC” or “isotype antibody”. In the left panel, exogenous EN2-RFP fusion protein gave off red fluorescence. In the middle panel, EN2 recognized by the monoclonal antibody gave off green fluorescence while no green fluorescence was detected when isotype antibody used as a negative control. Exogenous EN2-RFP fusion protein showed bright green while endogenous EN2 protein showed weak green stained with EN2 monoclonal antibody. Exogenous EN2-RFP fusion protein distributed in nucleus while the nucleus without exogenous EN2-RFP fusion protein showed as dark hole. The right panel was the merged images of left and middle panel. The sites with yellow color was the overlay of bright green and red color. The magnification of all image was 1000×. These experiments were repeated independently 3 times with similar results