CARD11 DI variants expressed at a 50:50 ratio to WT CARD11 disrupt CARD11-dependent signaling pathways to different extents. (A) Immunoblot analysis of JNK and NF-κB signaling in CARD11 KO Jurkat cells transiently transfected with a 50:50 ratio of WT CARD11-V5 and empty vector (EV), WT CARD11-FLAG, or DI CARD11-FLAG variants. Transfected cells were stimulated for 30 or 60 min with 20 ng/ml PMA and 1 μg/ml ionomycin (P/I). (B) Quantitation of the fold change of specified proteins over baseline from immunoblots in A; n = 3. (C and D) Flow cytometric analysis of CARD11 KO NF-κB-EGFP Jurkat cells transfected as in A and stimulated with 1 μg/ml anti-CD3/anti-CD28 antibodies or P/I for 6 h. (C) Quantitation of the mean fluorescent intensity (top) or %GFP⁺ cells (bottom; n = 3). (D) Representative histograms demonstrating the GFP⁺ gate (dashed lines) and %GFP⁺ cells (numbers) summarized in C. (E) Representative immunoblot from transfected cells in C and D. (F) Representative immunoblot from transfected cells in G and H. (G and H) Flow cytometric analysis of CARD11 KO Jurkat cells transfected as in A in addition to an AP-1–driven GFP reporter and stimulated with P/I for 6 h. (G) Quantitation of the %GFP⁺ cells (n = 5). (H) Representative histograms demonstrating the GFP⁺ gate (dashed lines) and %GFP⁺ cells (numbers) summarized in G. (I) Representative immunoblot confirming ectopic CARD11 expression in stably transduced Jurkat cells used in Fig. 3 I. Immunoblots are representative of at least three independent experiments. Error bars indicate the SEM. Asterisks denote significance by one-way ANOVA followed by Dunnett’s multiple comparisons (B) or two-way ANOVA followed by Šídák’s multiple comparisons (C and G). *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. EV, empty vector; MFI, mean fluorescence intensity; and NS/No Stim., no stimulus. Source data are available for this figure: SourceData FS2.