15d-PGJ2 inhibits NF-κB activity in HS-Sultan cells. HS-Sultan cells were treated with 15d-PGJ2 (10 μM) or control diluent. (A) At the indicated times, whole-cell extracts were analyzed for NF-κB activation by EMSA (left panel). A section of the fluorogram is shown. Positions of NF-κB-DNA (NF-κB) and nonspecific protein-DNA (ns) complexes are indicated. The levels of NF-κB DNA-binding activity in control (○) and 15d-PGJ2-treated (•) cells were quantified by MDP analysis and expressed as arbitrary units (right panel). (B) At 2 and 4 hours after 15d-PGJ2 treatment, levels of total (IκBα) and phosphorylated (P-IκBα) IκBα were determined by Western blot analysis. Levels of α-tubulin are shown as control. (C) In a parallel experiment, HS-Sultan cells were treated with 10 μM 15d-PGJ2 (•) or control diluent (○) for 2 hours and then stimulated with the phorbol ester TPA (100 ng/mL). At different times after treatment, whole-cell extracts were analyzed for NF-κB DNA-binding activity by EMSA, and the levels of NF-κB DNA-binding activity were quantitated by MDP analysis and expressed as arbitrary units. (D) HS-Sultan cells were treated with 15d-PGJ2 (10 μM) or control diluent for 2 hours prior to stimulation with TPA (100 ng/mL). At 15 minutes after TPA addition, whole-cell extracts were analyzed for IKK activity by kinase assay (KA: IKK). Endogenous IKK recovery was determined in the same samples by immunoblot analysis for IKKα (WB: IKKα).
