Figure 4.

PI3-kinase activity is necessary for NF-κB induction by pV. (A) Effect of wortmannin on NF-κB DNA-binding activity. Jurkat cells were pretreated for 1 h with the indicated concentrations of wortmannin (W) and were stimulated with 1 mM pV (lanes 2–7) or 50 ng⋅ml⁻¹ TNFα (lanes 8–13) for 1 h. Nuclear extracts were prepared and electrophoretic mobility shift assays were performed by using the IL-2 gene κB site as probe. The position of the NF-κB complex is indicated by an arrow. (B) Effect of wortmannin on the activation of a NF-κB-dependent reporter gene activity. Jurkat T cells were transiently transfected with a plasmid containing the luciferase reporter gene driven by three repeats of the HIV-1 κB enhancer. Forty-eight hours after transfection, cells were pretreated with 200 nM wortmannin for 1 h and then induced with 1 mM pV or 50 ng⋅ml⁻¹ TNFα for 6 h, as indicated. Luciferase activities were measured and normalized to the protein concentration in each extract. The values shown are averages (mean + SEM) of one representative experiment in which each transfection was performed in quadruplicate.