miR-144/451 disruption enhances the autophagic flux and proteasomal degradation of insoluble free α-globin. Reticulocytes in whole blood from mice with the indicated genotypes were pulse-labeled with ³⁵S-amino acids; chased with unlabeled amino acids ± a proteasome inhibitor (MG132 [MG]; 10 μM), a lysosome inhibitor (chloroquine [CQ]; 100 μM), or vehicle (Veh); separated via centrifugation into soluble and insoluble fractions; subjected to TAU gel electrophoresis to separate globin proteins; and analyzed by autoradiography. (A) Autoradiogram immediately after a 30-minute pulse with ³⁵S-amino acids. Each lane represents a biological replicate experiment using equivalent input cell numbers from different mice. (B) Relative levels of soluble and insoluble globin determined by quantitative image analysis of TAU gel autoradiograms, as shown in panel A. (C) Representative TAU gel autoradiograms showing soluble and insoluble globin chains after 3-hour and 6-hour chases with unlabeled amino acids. (D) Results of multiple experiments performed as described for panel C and quantified using automated image analysis. n = 5 mice for each genotype. (E) Percentage increases in the clearance of insoluble α-globin in Hbb^th3/+miR^−/− vs Hbb^th3/+miR^+/+ reticulocytes after a 6-hour chase with MG, CQ, or Veh. (F) Summary of data from panel E indicating the relative contributions of autophagy (CQ-inhibited) or proteasomal (MG-inhibited) degradation to the enhanced clearance of free α-globin conferred by miR-144/451 disruption. Data are shown as mean values ± SD; ∗∗∗∗P < .0001; ∗∗P < .01; ∗P < .05.