mTORC1 activity is enhanced in β-thalassemia and suppressed by miR-144/451 disruption in 8-week-old mice. (A) Western blot analysis of indicated proteins and phosphoproteins (p) in the erythroblast population Ery.B from the bone marrow of mice with the indicated genotypes. (B) Quantification of multiple western blot experiments depicted in panel A. n = 3 or 4 mice for each genotype. pS6 Ser235/236 and pS6k Thr389 levels were normalized to the levels of the respective total proteins. CAB39 was normalized to β-actin. (C) pThr172 AMPKα levels in embryonic day 14.5 (E14.5) fetal liver cells of the indicated genotype as measured using sandwich enzyme-linked immunosorbent assay and normalized to total cellular protein determined using the bicinchoninic acid assay. (D) Flow cytometry quantification of pS6 Ser235/236 in Ery.A, Ery.B, and Ery.C bone marrow erythroblasts. n = between 4 and 13 mice for each genotype. (E) In vivo protein synthesis rates in individual maturation stage–matched bone marrow erythroblasts, as determined via flow cytometry for O-propargyl-puromycin incorporated into nascent proteins during translation. n = between 4 and 7 mice for each genotype. (F) CD71 MFI in Ery.A and Ery.B bone marrow erythroblasts. n = 13 or 15 mice for each genotype. (G) Nonheme iron levels in splenic Ter119⁺ erythroblasts, normalized to total cellular protein. n = 3 or 4 mice for each genotype. Bar charts show data as mean values ± SD; ∗∗∗∗P < .0001; ∗∗P < .01; ∗P < .05; MFI, mean fluorescence intensity.