E127G and G116S CARD11 spontaneously aggregate and drive constitutive NF-κB activation. (A) Sanger dideoxy DNA sequencing confirmation of heterozygous missense mutations (A to G substitution or vice versa, designated as purine or “R”) in exon 5 of the CARD11 gene in P1, P2, and P3 (not in mother [M]) and exon 4 of CARD11 in P4 (not in control [Ctrl]). Amino acids are shown in single letter code. (B) Schematic representation of the CARD11 protein (Lenz et al., 2008a). The E127G and G116S amino acid changes within the CC region of CARD11 are denoted. (C) Ectopic expression of WT, E127G, or G116S CARD11 as N-terminal fluorescent fusion proteins (myc/Venus-CARD11) in BJAB B cells. BCL10 or MALT1 were visualized by immunofluorescence staining using Alexa Fluor 594–conjugated secondary Abs. Bars, 1 µm. (D) Plasmids encoding Flag-tagged WT, E127G, G116S, or other DLBCL-derived somatic, active mutant CARD11 were transfected into CARD11-deficient JPM50.6 cells containing a κB-driven GFP reporter gene, and GFP expression was analyzed by flow cytometry. Histograms (left) and MFI of GFP⁺ cells (middle) are shown, indicating the relative amount of NF-κB activity. Immunoblots confirming expression of Flag-CARD11 proteins are shown at right; β-actin served as a loading control. (E) BJAB B cells were transfected with a GFP expression plasmid plus Flag-tagged CARD11 constructs as listed in D. Flow cytometry of BJAB B cells transfected with a GFP expression plasmid plus Flag-tagged CARD11 constructs as listed in D and then stained with anti-CD83 was performed. Quantitated MFI of CD83 expression in GFP⁺ cells is shown (middle). Immunoblots confirming expression of CARD11 proteins are shown at right with a β-actin loading control. (C–E) Data are representative of three (C and E) or four (D) independent experiments.