Total protein expression of DARC does not change significantly during reticulocyte maturation. (A) Reticulocytes were purified from peripheral blood as described in “Study design” and stained with CD71 and TO (RNA). The flow cytometry dot plot shows 4 subpopulations of circulating reticulocytes in adult peripheral blood: CD71^high/TO^high (R1), CD71^low/TO^high (R2), CD71^neg/TO^high (R3), CD71^neg/TO^low (R4), and erythrocytes (E). (B-C) Quantification of the dot plot shown in panel A, after reticulocyte enrichment (B; N = 13), and normalized to the actual frequencies in adult peripheral blood (C; N = 13). (D) Reticulocyte subpopulations defined in panel A were sorted, and total protein lysate was subjected to SDS-polyacrylamide gel electrophoresis and western blotting. Blots were stained with anti-band 3 (top) and anti-DARC (clone 2C3; bottom). Note that DARC (calculated peptide mass, 35 kDa) presents as a diffuse pattern of bands as previously described. *a-Specific bands (supplemental Figure 1A).^8,19