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. 1998 Apr;18(4):2077–2088. doi: 10.1128/mcb.18.4.2077

FIG. 1.

FIG. 1

FIG. 1

FIG. 1

FIG. 1

(A) Purification of Rep-κB activity from Jurkat-T cells. Nuclear extracts (NE), flowthrough (Flow), and eluted fractions were tested for binding to the oligonucleotide SL350, which contains both κB elements in the human NF-κB2 promoter and flanking regions (positions −119 to −63). The specificity of binding was analyzed by adding 20- and 60-fold molar excesses of a homologous competitor (lanes 3 and 4). A and B indicate the positions of specific DNA binding complexes. Complex A was identified as Rep-κB activity. (B) Sequence requirements for Rep-κB binding. Competition experiments were performed with eluted fraction 11 (5 μl) and 20- to 60-fold molar excesses of the unlabeled competitor (Comp.) oligonucleotides shown in panel C. As a probe, the 32P-labeled oligonucleotide SL350 was used. −, no competitor added. (C) Sequences and Rep-κB binding abilities of different oligonucleotides derived from the first κB element in the NF-κB2 promoter. Only the positive strand of each oligonucleotide is shown. Lowercase letters represent added linker sequences; dashes indicate identity with the consensus (Consens.) sequence. (D) Rep-κB activity is recognized by an antibody directed against RBP-Jκ. Treatment of eluted fractions 11 and 12 (5 μl) with an RBP-Jκ-specific antibody (α-RBP) specifically abolished band A. Instead, a more slowly migrating complex (band C, lanes 2 and 4) appeared. Another DNA binding activity (complex B) was not affected by the antibody. The probe was a 32P-labeled oligonucleotide, SL332, containing the first κB element (positions −119 to −90) within the NF-κB2 promoter. −, control (antibody not added). (E) Rep-κB binds specifically to the RBP-Jκ site in the murine HES-1 promoter. Nuclear extracts and eluted fractions from Jurkat-T cells were analyzed for specific DNA binding activity, using the 32P labeled oligonucleotide HES-1 as a probe. This oligonucleotide contains the known RBP-Jκ consensus sequence 5′-CTGTGGGAAAGA-3′. As a competitor, a 50-fold molar excess of unlabeled oligonucleotide SL332 (lane 2) or HES-1 (lane 3) was used. Band A represents Rep-κB binding activity. −, control (competitor not added).