Fig 4.

Effect of JAB1/Csn5 on NF-κB signalling. (A) NF-κB reporter gene assays after knock-down of JAB1 by RNA-interference (siJAB1) or after ectopic expression of wild-type JAB1 or mutant JAB1 (JAB1-MPN mutant). Basal NF-κB activity is shown as mean values ± SD from triplicates. The right panel shows the efficacy of the siRNA construct for knock-down of the endogenous, as well as ectopically expressed JAB1 as compared to a scrambled control-siRNA (si-con.). (B) Cullin-1 does not interact with a JAB1-variant comprising a mutation in the proteolytic MPN⁺ domain responsible for deneddylation and de-ubiquitination activity. Flag-tagged Cullin-1 (Flag-Cul1) was coexpressed with myc-JAB1, or mutant myc-JAB1 (JAB1-MPN mutant) in 293 cells followed by anti-flag immunoprecipitation and immunoblot analysis of the myc-tag and Cullin-1 in immunoprecipitates (IP). The asterisk represents an unspecific band. (C) Reporter gene assay as in (A) including transfection with flag-IKK2 in the absence or presence of TNFα (50 ng/ml) for 6 hrs.