HSP27 and GATA-1 expression during erythroblast differentiation. (A) Scheme of the model used for human primary erythroblast differentiation. A representative microscopic image shows the structure of the differentiating erythroblasts at the indicated time points. (B) Human primary erythroblasts were induced to differentiate in the presence of IL-3, Epo with or without TGFβ. At the indicated times, the level of GATA-1 and HSP27 was determined by Western blot. HSC70 an HSP90 serve as loading control. (C) Differentiating cells were treated or not with the protein synthesis inhibitor CHX (5μM, 8 hours). Cells were harvested at indicated days, and lysates were blotted with the indicated antibodies. The data are representative of 3 independent experiments. (D) Human primary erythroblasts induced to differentiate in the presence of IL-3 and Epo were, at day 8, treated or not with the proteasome inhibitor MG132 (20μM, 5 hours), and nuclear/cytosolic GATA-1 and HSP27 expression was assessed by Western blotting. (E) Human K562 cells were induced to differentiate with hemin (40μM). At the indicated days, GATA-1 and HSP27 levels were assessed by Western blot. HSP90 serves as a loading control. (F) When indicated, K562 cells induced to differentiate by the presence of hemin were treated for 5 hours with the proteasome inhibitor MG132 (20μM). Nuclear/cytosolic GATA-1 and HSP27 expression was assessed by Western blot. HSC70 and lamin B serve as loading controls. (G) The proteasome activity was determined by the measurement of Suc-LLVY-AMC cleavage in the control lysates from hemin-treated K562 cells in the absence () or presence () of MG132 (20μM, 5 hours). a.u. indicates arbitrary units; bars, SD; n = 3.