Reduced free α-globin in Hbb^th3/+ mice with the Hbb diffuse haplotype. (A) Soluble and insoluble globins in RBCs determined by biochemical fractionation and triton acid urea (TAU) gel electrophoresis. Representative TAU gel images are shown in the left panels and the results of multiple experiments are summarized in the graph on the right. The y-axis represents relative staining intensities of insoluble α-globin on TAU gels, measured by automated image analysis and expressed in arbitrary units. Hbb^th3/+ sing, n = 5; Hbb^th3/+ diff, n = 6. (B) Transmission electron microscopy of flow cytometry–purified reticulocytes showing electron-dense, α-globin inclusions. Representative micrographs are shown in the 2 left panels. The scale bars represent 2 μm. The graph on the right shows the results of quantitative automated image analysis of multiple mice with relative levels of α-globin inclusions indicated on the y-axis using an arbitrary scale. Hbb^th3/+ sing, n = 6; Hbb^th3/+ diff, n = 5. (C) Hbb/Hba mRNA ratios in Ter119⁺ erythroblasts from bone marrow (BM, left) or spleen (right) of mice with the indicated genotypes, determined by RNA-sequencing analysis. Five mice were assessed per genotype. (D) Hbb/Hba mRNA ratios in EryA and EryB erythroblasts isolated from BM and reticulocytes (retic) isolated from peripheral blood, determined by RNA-sequencing analysis. Four mice were assessed per genotype. (E) Intronic Hbb/Hba RNA ratios in the samples described for panel D. All graphs show data as mean value ± SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ns, not significant; SD, standard deviation.