Inhibition of EpoR activation is not responsible for changes in proliferation and survival associated with the release of erythroid differentiation blockage. (A) Cells were grown either in the presence or absence of dox for 3 days (100 ng/mL) and then deprived in both serum and Epo for 4 hours (S) before being stimulated 5 minutes with 10 U/mL Epo and 5% serum (R). Cell lysates were subjected to immunoprecipitation with anti-EpoR antibodies and analyzed by Western blotting using antiphosphotyrosine antibodies (PY) followed by anti-EpoR antibodies (EPOR). A representative image for A2B and control cells is shown. (B) Western blot analysis of STAT5, ERK, and AKT phosphorylation as readout of signaling pathways activated downstream of the EpoR in cells treated for 2, 3, or 4 days with or without dox (100 ng/mL) in the presence of Epo (1 U/mL). S stands for starved cells that have been deprived from serum and Epo during 4 hours. Total protein extracts were subjected to immunoblotting with anti–phospho-AKT (specific for phosphorylated serine 473) or anti-AKT antibodies, with anti–phospho-ERK1/2 or anti-ERK1/2 and with anti–phospho-STAT5 (recognizing tyrosine 694–phosphorylated form) or anti-STAT5. For each cell type, analysis was performed at least 3 times with similar results. A representative image for A2B and control cells is shown.