Fig. 6
Interdomain surface interactions within the RIP2–CARD filament. a Cartoons illustrated the details of Ia:Ib, IIa:IIb, and IIIa:IIIb interactions in RIP2–CARD filament. Residues at the interface are shown in stick models. b NF-κB promoter activation by RIP2 and RIP2 single-point mutants. These RIP2 point mutations localized on the interaction interfaces and showed loss of function in terms of NF-κB promoter activation. c NF-κB promoter activation by RIP2, RIP2 single-point mutants E445R, R458E, and RIP2 double mutant E445R andR458E. The single mutants all showed loss of ability to activate NF-κB promoter, while the E445R and R458E double mutant partially restored NF-κB promoter activation. Error bar represents standard deviation values of three independent repeats. d NF-κB promoter activation by RIP2 and RIP2 mutants in HEK293T cells. RIP2 single-point mutants N449D and RIP2 double mutant N449R andD495R showed loss of function. While RIP2 single-point mutants N449R, D495R, and double mutant N449D and D495R showed higher NF-κB luciferase activities. Error bar represents standard deviation values of three independent repeats. e Expression levels of all RIP2 mutants are similar. HEK293T cells were transfected with wild-type RIP2 and RIP2 mutants. Cells were harvested 24 h post transfection. Levels of over-expressed wild-type RIP2 and RIP2 mutants were measured by anti-RIP2 antibody. Western blots were representative of three independent experiments. f Confocal microscope images of wild-type RIP2–GFP, RIP2(E445R)–GFP, RIP2(R458E)–GFP, and the double mutant RIP2(E445R–R458E)–GFP. The charge-reversal double mutant regained the puncta formation activity. Gray scale bar: 5 μm
