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. 2018 May 24;9:1136. doi: 10.3389/fimmu.2018.01136

Figure 5.

Figure 5

Functional conservation of Bcl10 and CARD–CC proteins. (A) Human MALT1-dependent NF-κB induction by different Bcl10 homologs. The indicated E-tagged Bcl10 homologs were expressed in MALT1-deficient HEK293T cells together with an NF-κB-dependent luciferase reporter expression plasmid and a constituitively expressed β-galactosidase reporter gene. Where indicated (+), human MALT1 was also co-expressed. Luciferase values were normalized against β-galactosidase and expressed as fold induction compared to samples not expressing Bcl10 (EV). Error bars represent 95% confidence intervals (Student’s t-distribution). The expression of E-tagged Bcl10 homologs and human MALT1 was revealed by Western blotting and detection with anti-E-tag or anti-MALT1 antibodies, respectively (lower part). Full-length Bcl10 is indicated by closed arrow heads and cleaved Bcl10 by open arrow heads. MALT1 is indicated by an asterisk. Experiments were repeated two times. (B) Bcl10/CARD-dependent MALT1-mediated NF-κB induction. Wild-type human Bcl10 and hybrid Bcl10 clones where residues 1–102 of human Bcl10 were replaced by the corresponding residues from C. gigas (CgN) or Nematostella vectensis (NvN) Bcl10 were expressed in MALT1-deficient HEK293T cells together with an NF-κB-dependent luciferase reporter expression plasmid and a constituitively expressed β-galactosidase reporter gene. Where indicated, (+) human MALT1 was also co-expressed. Luciferase values were normalized against β-galactosidase and expressed as fold induction compared to samples expressing human wild-type Bcl10 without MALT1. Experiments were repeated two times. (C) Yeast-2-hybrid assay demonstrating conserved interaction between both Nvn and human Bcl10 and type 1 paracaspases. Left panel represents growth on non-selective media and right panel selective growth (-LTHA medium), on which only a combination of bait and prey clones with interacting proteins can grow. As an independent readout, clones with strong bait–prey interactions also stain blue from X-gal. (D) Full-length and C-terminal deletion (ΔC) constructs of human CARD-9 and Nvn CARD–CC were expressed in MALT1-deficient HEK293T cells together with NF-κB-dependent luciferase reporter gene and constitutively expressed β-galactosidase reporter gene plasmids. Where indicated, (+) human MALT1 was also co-expressed. Luciferase values are normalized against β-galactosidase and expressed as fold induction compared to samples not expressing CARD-9 or CARD–CC (EV). Error bars represent 95% confidence intervals (Student’s t-distribution). The lower part of the panel shows the expression of full-length and ΔC-mutant CARD-9 and Nvn CARD–CC constructs, respectively, as revealed by Western blotting and development with anti-Flag antibodies. An aspecific Flag signal at 75 kDa is sometimes visible in the MALT1-deficient HEK293T cells. Experiments were repeated two times.