Systemic proteasome inhibition activates alternate α-globin detoxification pathways. (A) Real-time RT-PCR quantification of HSP mRNA expression in Ter119⁺ erythroblasts from wild-type or Th3/+ mice treated with vehicle (CTRL) or bortezomib (BOR). Expression is normalized to β-actin and Hprt mRNA levels. Relative expression between different experimental groups is shown with vehicle-treated wild-type mice assigned an arbitrary value of 1.0. Data are shown for Hspa1a (i), Hspa1b (ii), Hsp90aa1 (iii), and Hsp105 (iv); n = 4 mice/group. *P < .05; *P < .01; ***P < .001. (B) Purified BM Ter119⁺ cells from mice treated with bortezomib (BOR, 1.0 mg/kg for 14 days) or vehicle (CTRL) were analyzed by Western blotting for the autophagosome marker LC3b. Two forms of LC3b are indicated: the unmodified form (I) and the phosphatidylethanolamine-conjugated form (II), which indicates active autophagosomes. β-actin expression was examined as a loading control. Mean ± SEM LC3b-II signal normalized to β-actin is shown for 4 mice for each group, with wild-type control treated mice set at 1. (C) Reticulocytes from β-thalassemic (Th3/+) mice were labeled with ³⁵S-cysteine and ³⁵S-methionine and chased with unlabeled amino acids in the presence or absence of proteasome (MG132, 10μM) or lysosome (CQ, chloroquine, 100μM) inhibitors. Soluble and insoluble fractions were purified and analyzed for labeled α-globin by triton acetic acid urea gel electrophoresis, followed by autoradiography. (D) Quantification of autoradiographs from panel C; n = 3 mice. *P < .001 versus control; #P < .001 versus MG or CQ. Note that the drugs inhibit the loss of insoluble α-globin additively.