MALT1 scaffolding- and protease-dependent GEP by oncogenic CARD11. (A) Western blot analysis of MALT1 KO and MALT1/TRAF6 double-KO (dKO) BJAB cells expressing CARD11 L232LI. Effects of loss of MALT1 and/or TRAF6 on CYLD and Regnase-1 cleavage are shown. (B) Schematic depiction of MALT1B protein and domains. The MALT1 C453A PM and E795A TBM are shown. (C) Western blot analysis of MALT1 KO (left) and MALT1/TRAF6 dKO (right) BJAB expressing CARD11 L232LI reconstituted with mock vector, MALT1 WT, or mutants. Cleavage of MALT1 substrates CYLD and Regnase-1 and expression of NFKBID/IκBNS was determined. Unspecific bands are marked with an asterisk. (D) Quantification of ΔMFI of the 6 × NF-κB–EGFP reporter in MALT1 KO and MALT1/TRAF6 dKO BJAB cells reconstituted with mock vector, MALT1 WT, or mutant constructs. n = 4 replicates, all error bars depict the mean ± SD, ANOVA with Tukey multiple comparisons, ∗∗∗∗P < .0001. (E) Gene set variation analysis (GSVA) quantifying the effects of MALT1 WT and mutants on a published ABC DLBCL–derived NF-κB gene signature induced by CARD11 L232LI. (F) Heat map of differentially expressed genes in the MALT1 KO BJAB cells expressing CARD11 L232LI in dependency of reconstitution with mock vector, MALT1 WT, PM, TBM, or PM/TBM. Red denotes higher, blue denotes lower expression. n = 4 for each sample. (G) Signed P_adj plot comparing gene induction by MALT1 PM and TBM compared with MALT1 WT reconstituted MALT1 KO BJAB cells expressing CARD11 L232LI. Genes in the top-left and the bottom-right corner are strongly TRAF6 binding or MALT1-protease dependent, respectively. Annotated are the genes from the heat map in panel F. (H) Relative transcript expression analysis of ZC3H12A, NFKBID, and NFKBIZ in dependency of the indicated reconstituted MALT1 mutants and MALT1 WT in MALT1 KO BJAB cells expressing CARD11 L232LI by qRT-PCR. n = 4, all error bars depict the mean ± SD, ANOVA with Tukey multiple comparisons; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001.