Glut1 expression is maintained during reticulocyte maturation and is not released in exosomes. (A) Red blood cells were obtained from 5-day-old mice (t0,) and reticulocyte maturation was induced by ex vivo culture (t48) in the absence or presence of the proteasome inhibitors MG132 and lactacystine (lact) or the DMSO solvent as a control. The levels of Glut1 and transferrin receptor (TfR) were monitored in all cell samples by immunoblot analysis using specific polyclonal and monoclonal antibodies, respectively (left panels). Exosomes from the plasma of 5-day-old mice as well as from 48-hour ex vivo–differentiated reticulocyte cultures described in panel A were obtained by differential centrifugation. Glut1 and TfR levels were monitored by immunoblot. (B) Surface Glut1 and TfR expression was monitored in freshly isolated RBCs from 5-day-old mice as well as following the 48-hour ex vivo maturation in the absence (DMSO) or presence of the proteasome inhibitors indicated. The percentage of Glut1⁺ and TfR^hi cells is indicated in each histogram.