Supplementary Fig. 1 Kinetics of appearence of the faster migrating form of Bcl-10.

Transcription

1 α-cd3 + α-cd28: Time (min): n.s. Supplementary Fig. 1 Kinetics of appearence of the faster migrating form of. Immunoblot of lysates from Jurkat cells were incubated for the indicated times in the absence or presence of cross-linked anti-cd3 and anti-cd28 (α-cd3 + α-cd28). Black and white arrowheads indicate the migration of uncleaved and cleaved, respectively. A non-specific (n.s.) band served as a loading control. Cleaved was still detectable after 6 h of stimulation. Data are representative of three experiments.

2 Time (min): Control CHX IκBα Supplementary Fig. 2 Generation of the faster migrating species does not require new protein synthesis. Immunoblot of lysates of Jurkat cells pretreated for 30 min with 10 µg/ml cycloheximide (CHX) or corresponding solvent (DMSO, Control ) alone, and then stimulated with PMA and ionomycin for the indicated time in the continuous presence or absence of the inhibitor. Black and white arrowheads indicate the migration of uncleaved and cleaved, respectively. CHX-treatment did not affect generation of the faster migrating (cleaved) species, while it efficiently inhibited the re-synthesis of IκB. Data are representative of two experiments.

3 Time (min): 0 Mock CARMA1-CC p-iκb VSV-CARMA1 Supplementary Fig. 3 CARMA1 oligomerization is required for cleavage. Immunoblot of lysates from Jurkat cells lentivirally transduced with empty vector (Mock) or a VSVtagged expression construct for the CARMA1 coiled coil domain (CARMA1-CC). Cells were stimulated with α-cd3 + αcd28. Inhibition of CARMA1 oligomerization by the CARMA1CC construct impaired both IκBα phosphorylation and cleavage, while it did not affect Erk activation. Data are representative of two experiments.

4 Control Time (min): MG p-iκbα Tubulin Supplementary Fig. 4 Inhibition of the proteasome does not prevent cleavage. Immunoblot of lysates from Jurkat cells pretreated for 30 min with the proteasomal inhibitor MG132 or solvent (Control) and then stimulated in the continuous presence or absence of the inhibitor with PMA and ionomycin. Black and white arrowheads indicate the migration of uncleaved and cleaved, respectively. Inhibition of the proteasome by MG132 did not affect cleavage, while it efficiently stabilized p-iκbα. Data are representative of two experiments.

5 Stimulus Time (min) z-vad-fmk Caspase 8 α-cd3 + α-cd28 FasL p-iκbα PARP n.s. Supplementary Fig. 5 Inhibition of caspase proteolytic activity does not prevent cleavage. Immunoblot of lysates from Jurkat cells pretreated for 30 min with 50 µm z-vad-fmk or solvent control (DMSO, Control ), and then stimulated with α-cd3 + α-cd28 antibodies or hexameric recombinant FasL for the indicated times. A non-specific band (n.s.) served as a loading control. Pretreatment of the cells with z-vad-fmk had no effect on α-cd3 + α-cd28- induced cleavage, while it potently inhibited FasL-induced caspase-8 processing. One experiment was performed.

6 FLAG- VSV-MALT1 R228G + T229A V230A + + S231A + phosphoisoforms Anti- Supplementary Fig. 6 Mutation of R228, but not of T229, V230 or S231 affect MALT1-dependent cleavage in 293T cells. Immunoblot of cell lysates from 293T cells transfected with the indicated FLAG-tagged point mutants of, together with an expression vector for VSV-tagged wild-type MALT1 (+) or mock (-) plasmid to normalize transfection efficiency. Point mutation of R228 (R228G), but not of the indicated three subsequent amino acid residues (T229A, V230A, or S231A) impairs MALT1-dependent cleavage. Note that under conditions of overexpression in 293T cells, MALT1 predominantly cleaves the phosphorylated isoforms of. Data are representative of four experiments.

7 Fold induction : MALT1: WT Δcasp H415A C464A VSV-MALT1 : Transfected Endogenous Supplementary Fig. 7 The active MALT1 caspase-like domain is required for optimal NF-κB activation in 293T cells. Dual luciferase assay (graph, top) on lysates from 293T cells co-transfected with the indicated expression constructs, with renilla-luciferase and NF-κB-firefly luciferase constructs, and with a mock plasmid to normalize transfection efficiency; relative NF-κB activity was determined 24 hours later. Inactivation of the MALT1 catalytic activity by deletion of the caspase-like domain (deletion of amino acids ) or mutation of the active site residues C464 or H415 resulted in total NF-κB activation levels of approximately 50 % of the maximum level achieved with wild-type MALT1. The results are expressed as means + s.d. (n = 3). The immunoblots show the expression of the transfected VSV-MALT1 constructs (top) and of endogenous or FLAG-tagged (bottom). Data are representative of three experiments.

8 Time (min): Control z-vrpr-fmk p-iκbα p-jnk Erk Supplementary Fig. 8 The MALT1 inhibitor z-vrpr-fmk does not affect IKK, Erk or Jnk activation. Immunoblots of lysates from Jurkat cells preincubated for 30 min with 75 µm z-vrpr-fmk or solvent (DMSO, Control ), and then stimulated with PMA and ionomycin for the indicated times in the continuous presence or absence of z-vrpr-fmk. Compared to control cells, cells treated with z-vrpr-fmk showed no obvious difference in the stimulation-induced phosphorylation of IκBα, Erk and Jnk. Data are representative of three experiments.

9 Infection rate: WT R228G Flag- Supplementary Fig. 9 Expression levels of FLAG- constructs in Jurkat cell populations used in Figure 6e. Immunoblots of cells transduced with serially diluted lentiviruses expressing wild-type or mutant FLAG- through infection. Cell populations with similar expression levels (indicated by arrows) were chosen for the adhesion experiment shown in Figure 6e. Data are representative of four experiments.