Demonstration of complex formation of Tax protein and CBP/P300 coactivator in nuclear extract with the Fuc-T VIICRE-like element.

Nuclear extracts were analyzed in DNAP assay. (A) Detection of CREB-1 protein and the bZIP domain peptide in the Fuc-T VIICRE-like element protein complex. Nuclear extracts from Jurkat T cells (lanes 1-2) or the bZIP domain peptide (lanes 3-4) were incubated with the biotinylated wild-type oligonucleotide (lanes 1 and 3) or the mutated one (lanes 2 and 4), blotted with the anti–CREB-1 antibody reacting to the bZIP domain specifically. (B) Detection of Tax protein in the Fuc-T VII CRE-like element–protein complex. JPX-9 cells were treated with CdCl₂ (15 ng/mL) for 24 hours to induce Tax protein in vivo (lanes 2 and 4) and without CdCl₂ (lanes 1 and 3). Nuclear extracts were incubated with the biotinylated wild-type oligonucleotide (lanes 1-2 and 5-7) or the mutated oligonucleotide (lanes 3 and 4) of the CRE-like element, blotted with the anti-Tax antibody. Nuclear factor–dependent association of exogenous Tax was detected in the presence of histidine-Tax (lane 7). (C) Association of CBP/P300 coactivator in the nuclear protein–complex formation with Tax. Nuclear extracts of Jurkat T cells were incubated with the CRE-like element in the presence of inactive Tax mutant histidine–d3 (lane 7) or histidine–Tax (lane 8). DNA-protein complexes were analyzed by immunoblotting with anti-CBP. Mr indicates kilodaltons of molecular size markers; and ED, ED40515-N.