TNF-α activates JNK in HCD57 cells.

(A) Western blot analysis of HCD57 cells deprived of EPO for 18 hours prior to treatment. Cells were treated with nothing (lane 1), 100, 10, or 1 ng/mL TNF-α (lanes 2-4), or EPO (1 U/mL) (lane 5), and whole cell lysates were probed for anti–phospho-JNK1/2 (top panel), anti–phospho AKT and anti–phospho ERK1/2 (middle panel), and anti-JNK1 (bottom panel). (B) In vitro kinase assay of JNK1 immunoprecipitates using glutathione-S–transferase (GST)–cJun as a substrate from HCD57 cells treated with EPO in either the absence (lane 1) or presence of 0.1 or 1.0 μg/mL anti–TNF-α antibody (lanes 3, 4), 1.0 μg/mL anti–TNF-α antibody plus 100 ng TNF-α (lane 2), or goat IgG control (lane 5). Shown are 2 separate experiments to indicate reproducibility of the result. Phosphorylated GST-cJun (top panel) and total JNK1 (bottom panel) are indicated.