Fig. 5. TNF-α -induced CCL4 expression in human monocytic cells is suppressed by NF-κB inhibitors. THP-1 human monocytic cells were pretreated with NF-κB inhibitors (Triptolide 10µM or Resveratrol 15µM) for 1hr and then cells were treated with TNF-α (10ng/ml) for 24 hrs. Cells and supernatants were collected and CCL4 mRNA in cell lysates and CCL4 secreted protein expression in culture supernatants were determined using real-time RT-PCR and ELISA, respectively. The data show significant downmodulation of CCL4 (A) gene (Triptolide: P<0.0001; Resveratrol: P<0.0001) and (B) protein (Triptolide: P<0.0001; Resveratrol: P<0.0001) expression in cells that were pretreated with NF-κB inhibitors compared to mock-treated controls stimulated with TNF-α. (C) To analyze TNF-α-induced NF-κB phosphorylation, cells were incubated with TNF-α (10ng/ml) and cell samples were collected at 5, 10, 15, 30, 60, and 120 min. Mock treated cells served as negative control. Cell lysates in RIPA buffer were calibrated for protein concentration, resolved by SDS-PAGE and NF-κB phosphorylation was assessed by using specific antibodies against phosphorylated and total NF-κB. The blot shows that TNF-α treatment of human monocytic cells induced NF-κB phosphorylation at 5-10 min that reduced thereafter.