Biologic effects of CARD15 variants discovered in EOS patients. (A) MDP-independent and -dependent NF-κB transactivation by CARD15 variants discovered in EOS patients. HEK293T cells were cotransfected with 30 ng expression construct of a CARD15 variant together with the NF-κB reporter plasmid and measured for NF-κB activity after 12 hours' incubation with (▪) or without (□) 5 μg/mL MDP. Mock vector and the wild-type (WT) CARD15 were used as controls. Values represent the mean of normalized data (mock without MDP = 1) of triplicate cultures, and error bars indicate SD. Shown in 1 representative result of 3 independent experiments. Protein expression levels of CARD15 variants analyzed by Western blotting are shown in the top column. (B) MDP-independent and -dependent NF-κB transactivation by combinations of CARD15 variants mimicking the genotype of EOS patients. To reach a phenotype closer to the heterozygous CARD15 gene expression in case 1 to 8, 15 ng of each CARD15 mutant (R334W, H496L, T605P, N670K, and M513T) was cotransfected with the same amount of the wild-type CARD15 with (▪) or without (□)5 μg/mL MDP. For case 9, 15 ng of each D382E and A612T were cotransfected. Mock vector alone and the wild-type CARD15 alone were added as controls.