DARC-specific antibodies and DBP display significantly higher binding to CD71^high/TO^highreticulocytes compared with other reticulocyte subsets or erythrocytes. (A) Schematic representation of 7-transmembrane spanning topology of DARC. N-terminal extracellular domain contains binding site for 2C3 and Fy6 antibodies, which overlap with DBP domain, indicated in gray. The Fy3 binding domain is localized at the third extracellular loop outside the DBP binding pocket. (B) Fold change representation of binding of DARC-specific antibodies to the 4 reticulocyte subpopulations and erythrocytes, as defined in Figure 1A. Enriched fraction of reticulocytes was probed with anti-Fya, anti-Fyb, Fy3, 2C3, and Fy6 antibodies followed by staining with anti-CD71 and TO. Figure is depicted as a fold change normalized to erythrocytes (anti-Fy6; anti-2C3; anti-Fya; anti-Fyb; anti-Fy3; n ≥ 6). (C) Fold change representation CD235a (glycophorin A [GPA]) antibody binding to the different reticulocyte populations and erythrocytes (N = 3). (D) Enriched fraction of reticulocytes was incubated with biotinylated recombinant DBP and counterstained with anti-CD71 and TO. A representable histogram semioffset is depicted, and a quantification of the mean fluorescent intensity (MFI) of DBP-binding (N = 6). Student t test (paired) was used to calculate statistical significance (*P < .05; **P < .01; ***P < .001; ****P < .0001). (E) ImageStream images depicting erythrocytes (top 2 panels), TO^high/lowCD71^neg (middle), and TO^highCD71^high (bottom) stained with RNA (TO, green), CD71 (red), and DBP (purple) as well as normal light. (F) Binding of CD71⁺ reticulocytes on SRP-streptavidine biosensor. CD71⁺ reticulocytes were isolated as indicated in “Study design” and flowed over biosensor coated with DBP (1000 nM) and anti-GPA (n > 100). The response and sedimentation signals were measured and represented as normalized total response/sedimentation response (T/S). Columns and bars represent means and standard deviation, respectively. One-way analysis of variance was used to calculate statistical significance (*P < .05; **P < .01; ***P < .001; ****P < .0001). BSA, bovine serum albumin; IgG, immunoglobulin G.