Activation of MAPKs (JNK, ERK, and p38) is required for PS-F2-stimulated TNF-α production in macrophages. (A) RAW264.7 cells were untreated or stimulated with PS-F2 (8 μg/ml), and total cell lysates were prepared at different times after stimulation. Equal amounts of cell lysates from the samples were subjected to Western blotting with antibodies specific for phosphorylated and total JNK, ERK and p38. (B) Signals in panel A were quantified by densitometric analysis, and phospho-specific signals were normalized against total protein signals. Bars indicate the fold changes of phosphorylated MAPK amounts at indicated time points over those at 0 min (n = 3). (C) RAW264.7 cells were pre-incubated with or without UO126 (ERK inhibitor), SB202190 (p38 inhibitor), or SP600125 (JNK inhibitor) for 30 min and stimulated with PS-F2 (15 μg/ml) for additional 20 h in the presence or absence of inhibitors. Cells left untreated or treated with inhibitors alone served as controls. TNF-α levels in the culture fluids were determined by ELISA (n = 3). Data shown are representative of 3 or more experiments. *P < 0.05, **P < 0.01 versus PS-F2 stimulation alone in (C).