Figure 4

PG490 inhibited both NF-κB and AP-1 DNA-binding and transcriptional activity. T cells were pretreated with various concentrations of PG490 (A) for 2 h and then stimulated with TNF-α (B) or CD3/CD28 (C) for 6 h. The nuclear extracts were analyzed for both NF-κB and AP-1 DNA-binding activity by EMSA. In (D), T cells were pretreated with PG490 for 2 h or left untreated, and then stimulated with TNF-α for various lengths of time as indicated. After washing, cell pellets were collected and cytoplasmic extracts were analyzed for the protein levels of IκBα and β-actin by Western blotting. In (E), T cells were mixed together with pNF-κB-Luc or pAP-1-Luc reporter plasmids and the transfection procedures were performed as described for Figure 2. After electroporation for 48 h, the cells were aliquoted equally and pretreated with various concentrations of PG490 for 2 h. After stimulation with TNF-α for another 18 h, cells were collected and analyzed for luciferase activity. Representative data of at least 3 independent experiments are shown.