Improved ineffective erythropoiesis in double-heterozygote mice. (A) Morphology of RBCs in peripheral blood smears (n = 5 mice per group). (B) Deposition of α-globin on RBC membranes as assessed by analyzing proteins derived from RBC membranes using nondenaturing gel analysis. Data are representative of an experiment independently repeated five times. Standard “localization control,” RBC cytoplasmic lysate was run on the gel to identify where α- and β-globin migrate. (C) RBC survival in circulation from WT, TfR1^+/−, th3/+, and double-heterozygote mice as measured by the decaying percentage of biotinylated RBCs over time (n = 4 mice per group). (D) Double-heterozygote mice exhibit smaller spleen size in comparison with th3/+ mice. (E) Statistical analysis of spleen/body weight (n = 5-6 mice per group). (F) Ter119 immunohistochemistry staining reveals more normal splenic architecture with more surface area devoted to white pulp (Ter119 negative cells) in TfR1^+/− mice in relation to WT mice and in double-heterozygote mice in relation to th3/+ mice. In addition, Ter119 immunohistochemistry staining of liver sections reveals reversal of extramedullary erythropoiesis (circles) in double-heterozygote mice in relation to th3/+ mice (n = 4-6 mice per group). (G) Flow cytometry analysis of percentages of bone marrow erythroid precursors (red) and enucleated cells (black) from WT, TfR1^+/−, th3/+, and double-heterozygote mice as identified by CD44 and forward scatter^46,52  (n = 4-5 mice per group). (H) Serum erythropoietin concentration in WT, TfR1^+/−, th3/+, and double-heterozygote mice (n = 4-5 mice per group). *P < .05 vs WT mice; **P < .01 vs WT mice; ††P < .01 vs th3/+ mice. Double-het = double-heterozygote mice; FSC, forward scatter; NS, not significant; STD, standard.