SP600125 inhibits JNK activity. (A) An in vitro kinase assay of JNK1 immunoprecipitates isolated from HCD57 cells cultured in the presence of EPO alone (lane 1), EPO + SP600125 inhibitor (INH; lanes 2-6), or EPO + DMSO vehicle (lane 7) for 6 hours. Phosphorylated GST-cjun fusion protein (top), and total JNK1 (bottom) are indicated. (B) Graphic representation of inhibition of JNK activity by SP600125. HCD57 cells were cultured for 6 hours with EPO alone (–), EPO + SP600125 at the concentrations indicated, or EPO + DMSO vehicle (V). JNK activity is inhibited by approximately 90% by 12.5 μM inhibitor. (C-D) Graphic representation of effect of SP600125 on p38 (C) and ERK1/2 (D) phosphorylation. HCD57 cells were treated in EPO alone (–), EPO + DMSO vehicle (V), or EPO + 12.5 μM or 25 μM SP600125 for 6 hours. For ERK1/2 phosphorylation (D), HCD57 cells were deprived of EPO for 4 hours prior to treatment with SP600125 inhibitor for 30 minutes followed by stimulation with 10 U EPO/mL for 10 minutes. For panels C and D, phosphorylation is shown as percent phosphorylation compared with EPO treatment alone. Shown are the quantitative results of 2 independent phospho-p38 and ERK1/2 Western blots corrected against total p38 and ERK1/2 expression. Error bars represent standard error from the mean.