Figure 4

RNF121 is a broad regulator of NF-κB activation. (A) HEK293T cells stably expressing TLR3 were transfected with a control non-specific (NS) siRNA or with siRNAs against RNF121 (RNF121 a or b). 48 hrs later, the cells were also transfected with an NF-κB reporter. 24 hrs later, the cells were either left unstimulated or were stimulated with poly(I:C) (10 μg/ml) for 6 hrs and then were analyzed by luciferase assay with normalization against Renilla luciferase activity. The NS- and RNF121-silenced cells were also stimulated with poly(I:C) for the indicated times, then were subjected to immunoblotting analysis as indicated. (B) NS-,RNF121- and TRIF-silenced HEK293T stably expressing TL3 were transfected with an IFNβ reporter. 24 hrs later, the cells were either left unstimulated or were stimulated with poly(I:C) (10 μg/ml) for 6 hrs and then were analyzed by luciferase assay. (C) HEK293T cells stably expressing TLR4 were transfected as in (A). Then, cells were either left unstimulated or were stimulated with LPS (10 μg/ml) for 6 hrs and then were analyzed by luciferase assay. The NS- and RNF121-silenced cells were also stimulated with LPS (10 μg/ml) for the indicated times, then were subjected to immunoblotting analysis as indicated. (D) HEK293T cells were transfected as in (A). Then, cells were either left unstimulated or were infected with Sendai virus (SeV) for 6 hrs and then were analyzed by luciferase assay. HEK293T cells stably expressing NOD1 (E) or NOD2 (F) were transfected as in (A). Then, cells were either left unstimulated or were stimulated with IE-DAP or MDP (10 μg/ml) respectively for 6 hrs and then were analyzed by luciferase assay. (G) HEK293T cells were transfected as in (A). Then, cells were either left unstimulated or were treated with etoposide (VP16, 40 μm) for 6 hrs and then were analyzed by luciferase assay.