Figure 4.

MLN4924 inhibits LPS-induced proinflammatory cytokine production from neutrophils by suppressing NF-κB. (A) The purified peritoneal neutrophils were seeded into 24-well plates at a density of 5×10⁶ cells/well. Following pretreatment with 0.5 µM MLN4924 for 30 min, cells were stimulated with 100 ng/ml LPS for 0, 15 or 60 min. Cell lysates were then prepared to analyze the levels of neddylated Cullins, P-IKKα/β, P-IκBα, IKKα/β, IκBα and β-actin. Neutrophils were pretreated with 0.5 µM MLN4924 and/or 80 µM JSH-23 or an equal volume of DMSO for 30 min. Then neutrophils were treated with LPS for the indicated periods of time. The concentration of (B) IL-6 and (C) TNF-α in the supernatants was measured with ELISA. **P<0.01. LPS, lipopolysaccharide; NF-κB, nuclear factor κB; IκB, inhibitor of κB; P-IKK, phosphorylated IκB kinase; DMSO, dimethyl sulfoxide; IL, interleukin; TNF, tumor necrosis factor.