Requirements of TRAF6 and HOIL-1 for CBM and oncogenic CARD11 signaling in BJAB cells. (A,C) Western blot analysis of 3 HOIL-1 KO (A) and TRAF6 KO (C) clones after stimulation with P/I for the indicated time points to determine MALT1 substrate cleavage and NF-κB signaling. (B,D) Representative flow cytometric analyses (top panel) and quantification of changes in mean fluorescence intensity (ΔMFI, bottom panel) of the 6 × NF-κB–EGFP reporter in 3 HOIL-1 KO (B) and TRAF6 KO (D) BJAB cells after P/I stimulation (5 hours). n = 3 replicates, all error bars depict the mean ± standard deviation (SD), ordinary 1-way analysis of variance (ANOVA) with Tukey multiple comparisons, ∗∗∗P < .001. (E-F) Western blot analysis of overexpression of FS-CARD11 WT, L232LI, and L251P in HOIL-1 KO (E) and TRAF6 KO (F) BJAB cells to determine NF-κB signaling and MALT1 substrate cleavage. (G) Representative flow cytometric analyses (top panel) and quantification of ΔMFI (bottom panel) of the 6 × NF-κB–EGFP reporter in parental (left), HOIL-1 KO (middle), and TRAF6 KO (right) BJAB cells expressing CARD11 WT and mutants. N = 3 replicates, all error bars depict the mean ± SD, ordinary 1-way ANOVA with Tukey multiple comparisons; ns, not significant; ∗∗P < .01 and ∗∗∗∗P < .0001.