Analysis of band 3, RhAG, and GPA sorting during enucleation of WT and nb/nb erythroblasts. DIC and immunofluorescent micrographs of WT and nb/nb-enucleating erythroblasts, including nascent reticulocyte (R) and extruding nucleus (N); probed with rabbit anti–mouse GPC and Alexa Fluor 594–labeled goat anti–rabbit; IgG (red; A); probed with rat anti–mouse TER 119 and Alexa Fluor 488–conjugated donkey anti–rat IgG (green) or rabbit anti–mouse band 3 and Alexa Fluor 594–labeled goat anti–rabbit IgG (red; B); or probed with rat anti–mouse TER 119 and Alexa 488–conjugated donkey anti–rat IgG (green) or rabbit anti–mouse RhAG and Alexa Fluor 594–labeled goat anti–rabbit IgG (red; C). Nuclei were identified by 4′,6-diamidino-2-phenylindole staining (DAPI; blue). Dashed lines outline the spherical portion of extruding nuclei in the red and green images in which there is no fluorescent labeling of the nucleus. The number of enucleating erythroblasts examined under each staining condition was 6 or more. Of note, during extrusion the nucleus transiently deforms, and a portion of it is visualized within the nascent reticulocyte, as evidenced in these images.