The TRAF1 gene is a direct transcriptional target of p80HT. (A) Chromatin immunoprecipitation assay for binding of p80HT to the human TRAF1 promoter in vivo. Chromatin isolated from the indicated human cell lines was immunoprecipitated either with normal rabbit serum (IgG) or with anti-NF-κB2 serum and then amplified with human TRAF1 or telomerase reverse transcriptase promoter-specific primers. Input control indicates amplified total DNA. (B) Schematic representation of the human TRAF1 promoter structure with sequences of the κB1 and κB5 sites. (C) Electrophoretic mobility shift assay for TRAF1 κB5-binding activity in nuclear extracts of splenic lymphocytes from p80HT and wild-type (WT) mice. Two κB-binding complexes containing either NF-κB2 or NF-κB1 are indicated based on antibody-mediated supershift. The NF-κB2 complex was supershifted by anti-c-Rel or partially disrupted by anti-RelA. Preimmune rabbit IgG was used as control. (D) Reporter assays for luciferase expression under the control of wild-type, κB1-, or κB5-mutant TRAF1 promoter in HT1080 cells. Luciferase values were normalized to β-galactosidase activity to account for differences in the transfection efficiency. Bars represent means (± standard deviation) from 3 independent assays.