GATA-1S310 is phosphorylated in response to Epo stimulation of primary fetal liver and cultured erythroid cells. (A) Western blot analysis of nuclear extracts of MEL cells induced to differentiate in the presence or absence of 5 mM HMBA after 96 hours using anti-GATA1 (pS310), anti-GATA1 (N6), or anti-topoisomerase I (Topo I) antibodies (i). Benzidine staining of hemoglobin (ii) confirmed the undifferentiated and differentiated status of MEL cells used (i). (B-C) The Epo-dependent erythroleukemic HCD57 cells were Epo-starved overnight, and 5 × 10⁷ cells were stimulated with Epo (2 U/mL) for the indicated time points, in the presence or absence of inhibitors of PI3-kinase LY294002 (LY; 10 μM), Wortmannin (WO; 100 nM), inhibitor of ERK/MAP kinase PD98059 (PD; 50 μM), or inhibitor of p38 MAP kinase SB203580 (SB; 10 μM). Western blot analysis of nuclear extracts with the indicated antibodies. (D) Cells (from B-C) were stimulated with increasing doses of Epo for 1 hour, and nuclear extracts were subjected to Western blot analysis as in panel B. Results shown are representative of at least 3 independent experiments. (E) Fetal liver cells enriched for erythroid progenitors (TER 119^- cells) were starved overnight in vitro in serum-free media (SFEM; StemCell Technologies) in the presence of SF (50 ng/mL) and IL-6 (10 ng/mL) and stimulated or not with Epo (2 U/mL) for 2 hours. Nuclear extracts were subjected to Western blot analysis as in panel B.