CARD11 LOF variants do not reconstitute CARD11-dependent signaling pathways. (A) Schematic of 3x-FLAG–tagged CARD11 protein expressed from pUNO vector (signaling domains annotated). Approximate location of each tested LOF variant is denoted with a black arrow and the variant of interest. (B) Immunoblot analysis of JNK and NF-κB signaling in CARD11 KO Jurkat cells transiently transfected with empty vector (EV), WT, or LOF CARD11 variants and stimulated for 30 or 60 min with 20 ng/ml PMA and 1 μg/ml ionomycin (P/I). (C) Quantitation of the fold change of specified proteins over baseline from immunoblots in B; n = 3. (D and E) Flow cytometric analysis of CARD11 KO NF-κB-EGFP Jurkat cells transfected as in B and stimulated with 1 μg/ml anti-CD3/anti-CD28 antibodies or P/I for 6 h. (D) Quantitation of the mean fluorescent intensity (top) or %GFP⁺ cells (bottom; n = 3). (E) Representative histograms demonstrating the GFP⁺ gate (dashed lines) and %GFP⁺ cells (numbers) summarized in D. (F) Representative immunoblot from transfected cells in D and E. (G) Representative immunoblot from transfected cells in H and I. (H and I) Flow cytometric analysis of CARD11 KO Jurkat cells transfected as in B in addition to an AP-1–driven GFP reporter and stimulated with P/I for 6 h. (H) Quantitation of the %GFP⁺ cells (n = 3). (I) Representative histograms demonstrating the GFP⁺ gate (dashed lines) and %GFP⁺ cells (numbers) summarized in H. Immunoblots are representative of three independent experiments. Error bars indicate the SEM. Asterisks denote significance by two-way ANOVA followed by Šídák’s multiple comparisons test (D and H) or one-way ANOVA followed by Dunnett’s multiple comparisons test (C). *P < 0.05; **P < 0.01; and ****P < 0.0001. EV, empty vector; F, FLAG; L, LATCH; MFI, mean fluorescence intensity; NS/No stim., no stimulus. Source data are available for this figure: SourceData F2.