A non-sense mutation in the CARD11 gene is the cause of the T-cell activation defect. (A) Model of the CARD11 gene illustrating the site of the mutation in P1 and P2. (B) real-time PCR for the detection of CARD11 RNA isolated from bone marrow mononuclear cells. Amplicons spanning the 5′ and the 3′ ends of CARD11 cDNA. RAJI cells served as a positive control, NHDF as a negative control for CARD11 expression. HPRT cDNA amplification was used to prove cDNA integrity. (C) Immunoblot for detection of CARD11 proteins. Lysates of CARD11-deficient NHDF cells transfected with expression plasmids for WT or the mutant p.Cys150* CARD11 as well as Epstein-Barr virus lymphoblastic cell lines from P2 and a healthy donor were probed for CARD11. The antibodies recognized full-length protein (upper row) or the truncated CARD11 protein (third row). 20 µg protein was loaded for each lane except for NHDF/CARD11 WT (only 1 µg protein loaded). β-Actin was probed as loading control. (D) T cells of P2 and a healthy control were purified, transduced with WT CARD11 by retroviral transfection with pMX-CARD11-IRES-GFP, and tested for expression of IL-2 4 hours after stimulation with PMA/ionomycin. The gates were set on GFP^– cells (no transfection) and GFP⁺ cells (transfection). (E-F) IκB degradation and p65 phosphorylation in retrovirally transduced T cells was measured after stimulation with PMA/ionomycin for 15 minutes (black line). The gray area represents unstimulated cells. All transfection experiments were repeated at least twice with similar results.