Specific inhibition of constitutive NF-κB activity in HTLV-I–infected T-cell lines treated with Bay 11-7082.
(A) Nuclear proteins extracted from cultures of T-cell lines were examined for DNA binding by EMSA with a radiolabeled NF-κB–specific probe. All HTLV-I–infected T-cell lines (lanes 3-6) exhibited protein/DNA binding with complex formation, compared with uninfected negative control cell lines (lanes 1 and 2). Cold competition using 100-fold excess of unlabeled NF-κB, AP-1, or mutated NF-κB oligonucleotides demonstrated the specificity of the protein/DNA binding complex (lanes 7-9). (B) Inhibition of constitutive NF-κB activity in HTLV-I–infected T-cell lines. HTLV-I–infected T-cell lines placed in culture at 1 × 106 cells/mL were treated for 1 hour with 5 μM Bay 11-7082 and assessed for NF-κB binding. Additional studies in which each cell line was analyzed for up to 24 hours after treatment yielded the same results. (C) NF-κB subunit specificity was determined by using antibodies to the NF-κB components p50, p65, c-Rel, and p52, resulting in supershift. (D) Time course of NF-κB inhibition in SLB-1 cells treated with Bay 11-7082. SLB-1 cells were treated with 5 μM Bay 11-7082. After the indicated times, nuclear proteins were extracted and EMSA was performed. (E) Specific inhibition of NF-κB by Bay 11-7082. SLB-1 cells were treated with the indicated concentrations of Bay 11-7082. After 1 hour, nuclear proteins were extracted and EMSA was performed with NF-κB– or AP-1–specific radiolabeled oligonucleotide probes. The specificity of the NF-κB inhibition was maintained throughout the 24-hour assay in SLB-1 cells.
