RELT is able to activate NF-κB and binds TRAF1.

(A) Activation of NF-κB by RELT. The 293 cells were transfected with increasing doses of RELT, starting at 0.1 μg, 0.2 μg, 0.5 μg, 1.0 μg, and 2.0 μg of DNA. Vector and RELT cytoplasmic domain deletion mutant (RELT-cyt) were used at a concentration of 2.0 μg. All wells were transfected with 0.5 μg κB-Luc and 0.5 μg TK-βgal. The total amount of DNA transfected in all wells was 3 μg. All values are normalized to β-gal activity. The experiment is representative of 3 experiments. (B) RELT binds TRAF1. The 293 cells were transfected with 5 μg RELT-HA and 5 μg of either FLAG TRAF 1, 2, 3, 5, or 6. Cell lysates were immunoprecipitated (I.P.) with anti-FLAG beads, separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted (I.B.) with anti-HA HRP. Blots were then stripped and re-probed with anti-FLAG M2 to detect the presence of tagged proteins. (C) Activation of NF-κB by RELT is not inhibited by a dominant-negative form of TRAF2. The 293T cells were transfected with a total of 3 μg DNA. All wells were transfected with 0.25 μg κ_B-luc and 0.25 μg TK-βgal. Cells were also transfected with 2 μg of either vector, RELT, or HVEM and 0.5 μg TRAF2DN or vector. Transfections were performed in duplicates, and this experiment is representative of 2 experiments.