CARD11 KO leads to a loss of JNK and AP-1 activation. (A) Immunoblot analysis of CARD11-dependent and -independent pathways in WT or CARD11 KO Jurkat T cells stimulated with 20 ng/ml PMA and 1 μg/ml ionomycin (P/I) over time. (B) Immunoblot analysis of JNK and NF-κB signaling in WT or CARD11 KO Jurkat T cells stimulated for 30 min with P/I or 80 ng/ml anisomycin. (C and D) Flow cytometric analysis of WT or CARD11 KO Jurkat cells transfected with an AP-1–driven GFP reporter and stimulated with P/I for 6 h. (C) Quantitation of the %GFP⁺ in the absence or presence of 1 μM JNKi or 100 nM trametinib (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) Quantitation of the fold change in AP-1 family member DNA binding, normalized to unstimulated WT (NS) for each family member, in WT or CARD11 KO Jurkat T cells stimulated with P/I for 2 h (n = 4). All 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. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. EV, empty vector; NS/No stim., no stimulus. (G) Schematic detailing the effects of CARD11 KO on NF-κB activation, JNK1/2 phosphorylation/activation, and AP-1 activation. Created in BioRender. Bauman, B. (2025) https://biorender.com/s09c704. Source data are available for this figure: SourceData F1.