JNK is active but not EPO-dependent in primary erythroid cells. Western blot analysis of spleen cells isolated from phenylhydrazine-treated mice. (A) Cells were cultured in either the presence (lanes 2-5) or absence (lanes 6-9) of EPO for 15, 30, 45, or 60 minutes immediately after isolation of cells. (B) Cells were deprived of EPO for 3 hours and then cultured in 10 U EPO/mL for the times indicated. (C) Cells were deprived of EPO (lanes 2-4) or deprived of EPO for one hour and then stimulated with EPO (lanes 5-7) for the times indicated in the presence of 1% bovine serum albumin (BSA) (top), 2% (middle) or 20% (bottom) fetal calf serum (FCS) for the times indicated. Phosphospecific JNK (p-JNK1/2), ERK (p-ERK1/2), AKT (p-AKT), and total JNK1 (JNK1) are indicated.