Fig. 5: E3 ligase-dependent and -independent modulation of MALT1 substrate recognition by HOIP and TRAF6.

a–c Cleavage of MALT1 substrates HOIL-1 (a), CYLD (b) and Regnase-1 (c) in parental HOIP KO Jurkat T cells transduced with either mock vector, HOIP WT, HOIP C885S or HOIP ΔUBA by Western blot under unstimulated and P/I stimulated (30 min) conditions. Cleavage was quantified as a ratio of cleavage products to full-length protein from 3 biological replicates. d–f Cleavage of MALT1 substrates HOIL-1 (d), CYLD (e) and N4BP1 (f) in parental TRAF6 KO Jurkat T cells transduced with either mock vector, TRAF6 WT, TRAF6 R88A/F118A or TRAF6 C70A by Western blot under unstimulated and P/I stimulated (30 min) conditions. Cleavage was quantified as a ratio of cleavage products to full-length protein from 3 biological replicates. g Analysis of A20 cleavage in parental, TRAF6 KO, HOIP KO and TRAF6/HOIP DKO Jurkat T cells by Western blot, either untreated or after stimulation with P/I (30 min and 2 h). The non-specific (ns) band below A20 full length is indicated. A20 full-length amounts were normalized to expression level of β-Actin, and the ratio of cleavage product to full-length was determined from 4 biological replicates. h Western blot analyses of A20, HOIL-1 and CYLD cleavage in A20 KO Jurkat T cells reconstituted with A20 WT or ZnF4, ZnF7, ZnF4/7 mutants untreated or after P/I stimulation (1 h). The non-specific (ns) band below A20 full-length is indicated. The ratios of cleavage products to full-length were determined for 4 biological replicates. All bars represent the means ± SEM and p-values were calculated by one-way ANOVA (a–f, h) or two-way ANOVA (g) combined with Dunnett’s multiple comparisons test. Only significant p-values (<0.05) are shown. kDa kilodalton.