Ritonavir inhibits constitutive NF-κB activation. (A) HUT-102 cells treated with ritonavir (40 μM) for the indicated time were evaluated for NF-κB and AP-1 activation. (B) HUT-102 cells treated with the indicated concentration of ritonavir for 24 hours were evaluated for NF-κB and AP-1 activation. (C) Cold competition using 100-fold molar excess of unlabeled NF-κB and AP-1 oligonucleotides (lanes 2 and 3) demonstrated the specificity of the protein/DNA binding complexes. Specificity of NF-κB binding was also determined by using antibodies to the NF-κB components p50, p65, c-Rel, and p52, resulting in supershift (lanes 4-7). (D) HUT-102 cells were treated with the indicated concentration of ritonavir for 24 hours, and cell lysates were immunoblotted for IκBα (top) and phospho-IκBα (middle). Actin immunoblots confirm that similar amounts of cell extracts were analyzed (bottom). (E) Primary acute-type ATL cells treated with concentrations of ritonavir as indicated for 24 hours were evaluated for NF-κB and AP-1 activation. Where indicated, 100-fold excess amounts of competitor oligonucleotides were added to the reaction mixture (lanes 4 and 5). (F) The bands of phosphorylated IκΒα were down-regulated by ritonavir treatment. Detection of actin expression was used as an internal control.