Figure 5
Figure 5. Mapping and functional analysis of PLCG1 variants and CARD11 variants. (A) Schematic representation of PLCG1 protein with mapping of the 10 missense mutations identified in AITL (circles) or TFH-like PTCL (squares) cases. Previously described activating mutations are in green boldface and mutations previously described but not functionally tested are underlined. (B) Monitoring of PLCG1-mediated MALT1 activation via a FRET-based reporter assay. Data are represented as mean ± SEM from 3 independent experiments. Significant differences in activation activity were determined using 1-way ANOVA (**P ≤ .01; ***P ≤ .001). Representative western blot from a MALT1 activation experiment. PLCG1 expression is revealed by anti-Myc tag blotting, whereas MALT1 expression is shown by anti-MALT1 antibody. (C) NFAT luciferase reporter assay monitoring activity of PLCG1 mutants, compared with previously reported activating mutants (green). Data are represented as mean ± SEM from 7 independent experiments. Significant differences in activation activity were determined using 1-way ANOVA (*P ≤ .05; **P ≤ .01). Representative western blot from a luciferase assay experiment. Ectopic myc-tagged PLCG1 expression is revealed by anti-Myc. Anti-Actin blotting serves as loading control. (D) Schematic representation of CARD11 protein with mapping of the 3-point mutations found in 2 AITL (circles) and 1 TFH-like PTCL-NOS (square) patients. A previously described mutation is underlined. (E) Monitoring of CARD11-mediated MALT1 activation via a FRET-based reporter assay. Data are represented as mean ± SEM from 6 independent experiments. Significant differences in activation activity were determined using 1-way ANOVA (*P ≤ .05; **P ≤ .01). The known activating L244P variant (green) was used as a positive control for the experiment. Representative western blot from a MALT1 activation experiment. CARD11 expression is revealed by anti-HA tag blotting, whereas MALT1 expression is shown by anti-MALT1 antibody. (F) NF-κB luciferase reporter assay in Jurkat cells deficient for CARD11 monitoring activity of CARD11 mutants, compared with previously reported activating mutants (green). Data are represented as mean ± SEM from 4 independent experiments. Significant differences in activation activity were determined using 2-way ANOVA (*P ≤ .05; **P ≤ .01; ***P ≤ .001 compared with WT PMA/IONO).

Mapping and functional analysis of PLCG1 variants and CARD11 variants. (A) Schematic representation of PLCG1 protein with mapping of the 10 missense mutations identified in AITL (circles) or TFH-like PTCL (squares) cases. Previously described activating mutations are in green boldface and mutations previously described but not functionally tested are underlined. (B) Monitoring of PLCG1-mediated MALT1 activation via a FRET-based reporter assay. Data are represented as mean ± SEM from 3 independent experiments. Significant differences in activation activity were determined using 1-way ANOVA (**P ≤ .01; ***P ≤ .001). Representative western blot from a MALT1 activation experiment. PLCG1 expression is revealed by anti-Myc tag blotting, whereas MALT1 expression is shown by anti-MALT1 antibody. (C) NFAT luciferase reporter assay monitoring activity of PLCG1 mutants, compared with previously reported activating mutants (green). Data are represented as mean ± SEM from 7 independent experiments. Significant differences in activation activity were determined using 1-way ANOVA (*P ≤ .05; **P ≤ .01). Representative western blot from a luciferase assay experiment. Ectopic myc-tagged PLCG1 expression is revealed by anti-Myc. Anti-Actin blotting serves as loading control. (D) Schematic representation of CARD11 protein with mapping of the 3-point mutations found in 2 AITL (circles) and 1 TFH-like PTCL-NOS (square) patients. A previously described mutation is underlined. (E) Monitoring of CARD11-mediated MALT1 activation via a FRET-based reporter assay. Data are represented as mean ± SEM from 6 independent experiments. Significant differences in activation activity were determined using 1-way ANOVA (*P ≤ .05; **P ≤ .01). The known activating L244P variant (green) was used as a positive control for the experiment. Representative western blot from a MALT1 activation experiment. CARD11 expression is revealed by anti-HA tag blotting, whereas MALT1 expression is shown by anti-MALT1 antibody. (F) NF-κB luciferase reporter assay in Jurkat cells deficient for CARD11 monitoring activity of CARD11 mutants, compared with previously reported activating mutants (green). Data are represented as mean ± SEM from 4 independent experiments. Significant differences in activation activity were determined using 2-way ANOVA (*P ≤ .05; **P ≤ .01; ***P ≤ .001 compared with WT PMA/IONO).

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