Erythroid protein distribution in sorted populations of reticulocytes and extruded nuclei.

<p>Sorted populations of extruded nuclei and reticulocytes were lysed and either 5×10⁵ or 1×10⁶ reticulocytes and nuclei were loaded depending on the protein expression levels or antibody sensitivity. Western blotting was conducted on A) membrane proteins using a mouse monoclonal antibody to Band 3, rabbit polyclonals to RhAG, Rh, Flotillin-2, Glut1, GPC, CD44 and stomatin and a rabbit monoclonal to beta 1 integrin B) cytoskeletal proteins using mouse monoclonal antibodies to alpha spectrin, beta spectrin, ankyrin, protein 4.2 and actin and rabbit polyclonals to alpha adducin, protein 4.1 and p55. C) nuclear protein Lamin B using a goat polyclonal. D) ER protein calnexin using a monoclonal antibody. Blots for RhAG, Rh, band 3, GPC, CD44, alpha spectrin, beta spectrin, ankyrin, protein 4.2 and lamin B are representative of 3–4 repeats from 3–4 independent cultures and sorting experiments. Blots for flotillin-2, Glut1, stomatin, beta 1 integrin, alpha adducin, protein 4.1, p55, actin and calnexin are representative of 2 repeats from 2 independent cultures and sorting experiments. All western blots shown were conducted on material isolated from the same reticulocyte and nuclei sorting experiment.