Nuclear proteins such as histones are exported into the cytoplasm during normal erythroid differentiation. (A) Silver stain of nuclei isolated from cultured erythroblasts across differentiation (from left, lanes are control shRNA at 48 hours, blank, extruded nuclei, blank, and Xpo7-KD nuclei at 48 hours). Protein sizes in kilodaltons are shown on right. Mass spectrometry of each lane was performed; a complete dataset uploaded to the proteomics identification database (PRIDE) is at http://www.ebi.ac.uk/pride under accession no. 1-20140306-107700. (B) Immunofluorescence (IF) of mouse tissues (fetal liver and adult spleen) isolated ex vivo from C57/Bl6 mice showing migration of histones into the reticulocyte before and during enucleation. Red staining is against either histone 3 or histone 2A using Alexa-594–conjugated secondary antibody, and blue staining is against DNA using DAPI. Profiles on right reflect intensities of each channel (corresponding colors) across the axis of the white arrow shown in the merge micrograph. In all panels, scale bar = 10 μm. (C) Erythroblasts cultured in erythropoietin-containing media were harvested after 24, 36, and 48 hours and fractionated into cytoplasmic and nuclear extracts using the PARIS Isolation Kit (Life Technologies). Immunoblotting was performed against a cytoplasmic protein (glyceraldehyde-3-phosphate dehydrogenase), nuclear membrane protein (Lamin B), and histones H3 and H2A. S, short exposure of blot, 5 seconds; L, long exposure of blot, 15 minutes. (D) IF of cultured erythroblasts at 48 hours after culture showing migration of histones into the cytoplasm before and during enucleation. Three primary antibodies against nuclear proteins are shown: histone 1, macro histone H2A.1, and pan-methyl histone H3 on lysine 9; blue staining is against DNA using DAPI. Secondary antibodies used were Alexa-594–conjugated secondary antibody (macro H2A.1 and pan-methyl H3K9) or Alexa-488–conjugated secondary antibody (H1). In all panels, scale bar = 10 μm.