a, b) Quantification of %κB-GFP + JPM50.6 cells transfected in Figure 2a-d. Data are mean ± s.d. for 8 ( a) and 5 (b) separate experiments. Asterisks denote statistically significant differences (Student’s t-test) for each LOF mutant versus wild-type (WT) with (black) or without (gray) stimulation, P < 0.05. ( c) Flow cytometric histograms measuring NF-κB-driven GFP reporter expression in JPM50.6 cells transfected with empty vector (EV), WT, or Q945X CARD11 constructs, ± 24 h anti-CD3/CD28 stimulation. ( d) Quantification of NF-κB-driven GFP reporter expression (MFI) in transfected JPM50.6 cells. Data are mean ± s.e.m. for three separate experiments. ( e) Cropped immunoblot for CARD11-FLAG expression in transfected JPM50.6 lysates ( c). Data representative of three independent experiments. ( f) Flow cytometric histograms for JPM50.6 cells transfected with WT CARD11 plus EV, WT or mutant constructs and stimulated as in c. ( g) GFP MFI quantification for JPM50.6 cells transfected in g. Data are mean ± s.e.m. (right) for five separate experiments; asterisks denote significance versus stimulated WT+WT (E57D P = 1.4 x 10 −3; L194P P = 2.4 x 10 −3; Q945X P = 0.113). ( h) Cropped immunoblot for CARD11 expression in transfected JPM50.6 lysates described in h. Data representative of three independent experiments. ( i) NF-κB-driven luciferase activity in WT Jurkat cells transfected with CARD11 (EV, WT, R975W, Q945X) plus luciferase reporter plasmids. Data represent mean ± s.d. fold change in κB-driven luciferase activity ± 24 h stimulation, normalized to Renilla luciferase activity for three separate experiments. ( J) Quantification of % phospho-S6 + Jurkat cells transfected in Figure 2i,j. Data are mean ± s.d. for four separate experiments. Asterisks denote significance for each LOF mutant versus WT with stimulation (E57D P = 4.9 x 10 −3; L194P P = 5.2 x 10 −3; R975W P = 2.9 x 10 −3; dup183_196 P = 0.013). ( k) Percent inhibition of pS6 signal calculated for each mutant versus EV (gray) or WT (black) transfected cells in b, based on the change in % pS6 + cells ± stimulation. ( l) Flow cytometric histograms measuring phospho-S6 in Jurkat cells transfected with EV, WT, L194P or Q945X CARD11 constructs, ± anti-CD3/CD28 stimulation for 20 min. Data are representative of three independent experiments.