Figure 2.
Homozygosity for a LOF TBK1 mutation in the patient. (A) Family pedigree showing segregation of the TBK1 mutation. PCR products were amplified from genomic DNA extracted from the granulocytes of P1 and both parents and subjected to Sanger sequencing. (B)TBK1 mRNA levels (upper panel) were determined by RT-qPCR on HEK293T cells 24 and 48 h after transfection with empty vector (EV), WT, and mutant TBK1 constructs. Western blot analysis was performed to assess the levels of protein for TBK1 (lower panel), autophosphorylated TBK1 (p-TBK1, Ser172), IRF3, and autophosphorylated IRF3 (p-IRF3, Ser396) in HEK293T cells at 24 and 48 h after transfection with EV, N-terminally flag-tagged WT, and mutant TBK1 constructs. The results shown are representative of three independent experiments. (C) Pulse-chase analysis of WT and mutant TBK1 protein stability. HEK293T cells were transfected with flag-tagged WT or mutant TBK1 expression plasmids for 24 h. Cells were then treated with cycloheximide (CHX; 100 ng/ml) for the indicated time points to inhibit protein synthesis, followed by western blot analysis (bottom). TBK1 protein levels were quantified by densitometry, normalized to GAPDH, and plotted over time (top). Data shown are representative of three independent experiments. The data shown are the means ± SEM of three independent experiments. P values were obtained by one-way ANOVA with Tukey’s multiple comparisons tests, and the P values for the 4-, 8-, and 12-h time points are indicated for the comparison of P1’s cells with control cells. ns; P > 0.05; *P < 0.05; and **P < 0.01. (D) ISRE, IRF3, and NF-κB promoter-driven luciferase reporter assays were performed on HEK293T cells 24 h after transfection with ISRE, IRF3, or NF-κB reporter plasmids along with EV, WT, and mutant TBK1 constructs. Luciferase activity was measured to assess TBK1-mediated activation. The results shown are representative of three independent experiments. The data shown are the means ± SEM of three experiments with three biological replicates, P values were obtained by one-way ANOVA with Tukey’s multiple comparisons tests. ns; P > 0.05, ****P < 0.0001. Source data are available for this figure: SourceData F2.

Homozygosity for a LOF TBK1 mutation in the patient. (A) Family pedigree showing segregation of the TBK1 mutation. PCR products were amplified from genomic DNA extracted from the granulocytes of P1 and both parents and subjected to Sanger sequencing. (B)TBK1 mRNA levels (upper panel) were determined by RT-qPCR on HEK293T cells 24 and 48 h after transfection with empty vector (EV), WT, and mutant TBK1 constructs. Western blot analysis was performed to assess the levels of protein for TBK1 (lower panel), autophosphorylated TBK1 (p-TBK1, Ser172), IRF3, and autophosphorylated IRF3 (p-IRF3, Ser396) in HEK293T cells at 24 and 48 h after transfection with EV, N-terminally flag-tagged WT, and mutant TBK1 constructs. The results shown are representative of three independent experiments. (C) Pulse-chase analysis of WT and mutant TBK1 protein stability. HEK293T cells were transfected with flag-tagged WT or mutant TBK1 expression plasmids for 24 h. Cells were then treated with cycloheximide (CHX; 100 ng/ml) for the indicated time points to inhibit protein synthesis, followed by western blot analysis (bottom). TBK1 protein levels were quantified by densitometry, normalized to GAPDH, and plotted over time (top). Data shown are representative of three independent experiments. The data shown are the means ± SEM of three independent experiments. P values were obtained by one-way ANOVA with Tukey’s multiple comparisons tests, and the P values for the 4-, 8-, and 12-h time points are indicated for the comparison of P1’s cells with control cells. ns; P > 0.05; *P < 0.05; and **P < 0.01. (D) ISRE, IRF3, and NF-κB promoter-driven luciferase reporter assays were performed on HEK293T cells 24 h after transfection with ISRE, IRF3, or NF-κB reporter plasmids along with EV, WT, and mutant TBK1 constructs. Luciferase activity was measured to assess TBK1-mediated activation. The results shown are representative of three independent experiments. The data shown are the means ± SEM of three experiments with three biological replicates, P values were obtained by one-way ANOVA with Tukey’s multiple comparisons tests. ns; P > 0.05, ****P < 0.0001. Source data are available for this figure: SourceData F2.

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