Supplementary Figure 1 : Atopy-associated CARD11 mutants reduce the percentage of transfected cells signaling to NF-κB and mTORC1, and the Q945X mutant is functionally “null” and does not interfere with wild-type CARD11 signaling.

From: Germline hypomorphic CARD11 mutations in severe atopic disease

Supplementary Figure 1

(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.