Fig. 3. NF-κB2 (P52) exhibits major inhibitory activity on the CD99 promoter. (A) p52 alone or in combination with RelA, c-Rel, RelB, and p65/c-Rel was transfected into 293T cells for the repression assay. (B) Mouse embryonic fibroblasts from wild-type control mice, and NF-κB2 precursor (p100/p52) - or NF-κB1 precursor (p105/ p50)-deficient mice were transfected with LMP1. (C) MEFs from wild-type control, IKKα-/-, IKKβ-/-, and IKKγ-/- mice were transiently transfected. In (A-C), cells were transiently co-transfected with the -1643~+123-Luc CD99 promoter and pGKβ-galactosidase control reporter, and subjected to reporter assays and Western blotting 20 h (A) or 48 h after transfection, using equal amounts of protein extracts (B). The p52 heteromeric complexes with all other subunits exhibited significant repressive activity in a dose-dependent manner, whereas the p50 heteromeric complexes had no or a much lesser inhibitory effect (Supplementary Fig. 3). (D) LMP1 stimulates p100 processing to p52. Approximately 105 293T cells were transfected with increasing amounts (0, 0.03, 0.1, 0.3, and 1 μg) of pCDNALMP1, RSV-p52 (0.1 μg), or NIK (0.1 μg) as controls. At posttransfection day 2, cell lysates were separated and transferred to nitrocellulose before being probed with the anti-p52 mouse monoclonal antibody (C-5, Santa Cruz). The processing of p100 by LMP1 results in 3 major bands, likely terminating at aa 445 (not fully processed, upper band) or aa 405 (lower band, p52).
