NF-κB activation 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 the cytosolic and nuclear fractions were prepared at indicated time points. Equal amounts of protein from the cytosolic fractions were subjected to Western blotting for I-κB and actin, and equal amounts of protein from the nuclear fractions were subjected to Western blotting for NF-κB and HDAC1. (B) Signals in panel A were quantified by densitometric analysis. I-κB signals were normalized against actin signals, and NF-κB signals were normalized against HDAC1 signals. Bars indicate the fold changes of I-κB and NF-κB amounts at indicated time points over those at 0 min (n = 3). (C) RAW264.7 cells were pre-incubated with or without MG132 or 481408 (NF-κB activation inhibitors) 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 < 0.01 versus PS-F2 stimulation alone in (C).