Somatic second-site mutation restores CARD11 expression and NF-κB signaling in patient T cells and Jurkat cell line. CARD11-deficient JPM50.6 cells were reconstituted with the indicated alleles of CARD11 by retroviral transduction with pMX-CARD11-IRES-GFP. GFP⁺ cells were FACS-sorted and expanded. (A) Immunoblot analysis of CARD11 expression compared with expression of CARD11 in Jurkat cell lines and nontransduced JPM50.6 cells. The blot is representative of 2 independent experiments. (B) Phosphorylation of p65 after stimulation with PMA/ionomycin (black line). The shaded area represents unstimulated cells. The assay was repeated 3 times with similar results. (C) Jurkat, JPM50.6, and CARD11-transduced JPM50.6 cells were transiently transfected with a firefly reporter gene construct responsive to NF-κB and a Renilla reporter under control of a CMV promotor for normalization. Bars represent firefly activity normalized to Renilla activity after stimulation with PMA/ionomycin (black bars) and without stimulation (white bars). Pooled data of 3 independent experiments are shown.