Role of Syk activation in PS-F2-stimulated TNF-α production in macrophages. (A) RAW264.7 cells were untreated or treated with piceatannol (10 and 25 μM) for 1 h and stimulated with PS-F2 (10 μg/ml) or poly (I:C) (10 μg/ml) for additional 20 h. Cells left unstimulated or stimulated with poly (I:C) served as controls. TNF-α concentrations in the culture fluids were determined by ELISA (n = 3). Data shown are representative of 3 or more experiments. (B) RAW264.7 cells were pre-incubated with or without piceatannol (25 μM) for 1 h and stimulated with PS-F2 (8 μg/ml) in the presence or absence of piceatannol. Cells left untreated served as controls. At 30 min after stimulation, cytosolic fractions and total cell lysates were prepared. Equal amounts of protein from the cytosolic fractions were subjected to Western blotting for I-κB and actin, and equal amounts of total cell lysates were subjected to Western blotting for phosphorylated and total JNK, ERK and p38. Changes in the I-κB signals (normalized against actin signals) and the phospho-specific signals (normalized against the total protein signals) are expressed as fold changes over the untreated group signals and indicated below each lane. * P < 0.05, **P < 0.01 versus PS-F2 stimulation alone in (A).