κB2 of the BAX promoter is necessary for the inhibition of BAX promoter activity by LMP-1. (A) EMSA with κB1, κB2, and κB3 wild-type and mutant oligonucleotides as probes without (-) or with nuclear extracts from 1852.4 cells grown 6 days in the absence (Tet-) or presence (Tet+) of 2 mg/mL tetracycline. LMP-1-dependent complexes are indicated by black arrows. An LMP-1-independent complex is indicated by a white arrow. (B) EMSA competition experiment with nuclear extracts of 1852.4 grown in the presence of tetracycline and radiolabeled wild-type κB1, κB2, and κB3 oligos and an excess unlabeled κB1, κB2, κB3, or κB consensus region (κBc) wild-type (w) or mutant (m) oligonucleotides. (C) EMSA supershift analysis with κB1, κB2, and κB3 oligonucleotides as probes and nuclear extracts of 1852.4 cells grown in the presence of tetracycline. Antibodies to p65, p50, and p52 were added to the binding reactions before incubation with the probe. Supershifted complexes are indicated by white (p65) and black (p50) arrows. (D) CAT assay of HEK 293-T cells cotransfected with reporter plasmids with wild-type (pBAX-CAT) or mutant BAX promoters (pBAX-CATm-κB2 or pBAX-CATm-κB3) and with pHEBo (□) or pLMP-1 (▪). (E) CAT assay of HEK 293-T cells transfected with either pBAX-CAT, pBAX-CATm-κB2, or pBAX-CATm-κB3 in combination with either pCMV-T (□), pCMV-T-p50 (▨), or pCMV-T-p65 together with pCMV-T-p50 (▪). (A-C) Representative example of 3 independent experiments. (D-E) CAT activity in the presence of the control vector was set to 100%. Mean and standard deviation of 3 independent experiments. ^*P < .01.