DBR1 mutants from the patients fail to potentiate the PKR-mediated stress response. (A) WT DBR1 or DBR1 variants from patients with brainstem encephalitis were overexpressed in HEK293T cells and then stimulated with polyI:C transfection (1 µg/ml). The cells were lysed and subjected to WB for detection of the indicated proteins. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (B and C) SV40-transformed fibroblasts (SV40-fibroblasts) from control and two patients, P1-DBR1I120T/I120T (B) and P2-DBR1Y17H/Y17H without and with WT DBR1 rescue (C) were transfected with 1 µg/ml polyI:C and subjected to WB with the indicated antibodies. Tubulin was used as a loading control. Data shown are representative of three independent experiments. (D) SV40-fibroblasts from control or DBR1-deficient patients were infected with VSV, and RT-qPCR was performed to assess the expression of the indicated ISR genes. Data are representative of three independent experiments. Graphs depict the mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ***, P < 0.001; ****, P < 0.0001. (E) SV40-fibroblasts from three patients, P1-DBR1I120T/I120T, P2-DBR1Y17H/Y17H, and P3-DBR1Y17H/Y17H without or with WT DBR1 rescue were infected with VSV at an MOI of 0.1, followed by WB for indicated antibodies. Tubulin was used as a loading control. Data shown are representative of three independent experiments. (F) SV40-fibroblasts from a healthy control and a DBR1-deficient patient were infected with HSV-1 at an MOI of 0.5, followed by WB for indicated antibodies. Tubulin was used as a loading control. Data shown are representative of three independent experiments. Source data are available for this figure: SourceData F2.
Figure 2.

DBR1 mutants from the patients fail to potentiate the PKR-mediated stress response. (A) WT DBR1 or DBR1 variants from patients with brainstem encephalitis were overexpressed in HEK293T cells and then stimulated with polyI:C transfection (1 µg/ml). The cells were lysed and subjected to WB for detection of the indicated proteins. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (B and C) SV40-transformed fibroblasts (SV40-fibroblasts) from control and two patients, P1-DBR1I120T/I120T (B) and P2-DBR1Y17H/Y17H without and with WT DBR1 rescue (C) were transfected with 1 µg/ml polyI:C and subjected to WB with the indicated antibodies. Tubulin was used as a loading control. Data shown are representative of three independent experiments. (D) SV40-fibroblasts from control or DBR1-deficient patients were infected with VSV, and RT-qPCR was performed to assess the expression of the indicated ISR genes. Data are representative of three independent experiments. Graphs depict the mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ***, P < 0.001; ****, P < 0.0001. (E) SV40-fibroblasts from three patients, P1-DBR1I120T/I120T, P2-DBR1Y17H/Y17H, and P3-DBR1Y17H/Y17H without or with WT DBR1 rescue were infected with VSV at an MOI of 0.1, followed by WB for indicated antibodies. Tubulin was used as a loading control. Data shown are representative of three independent experiments. (F) SV40-fibroblasts from a healthy control and a DBR1-deficient patient were infected with HSV-1 at an MOI of 0.5, followed by WB for indicated antibodies. Tubulin was used as a loading control. Data shown are representative of three independent experiments. Source data are available for this figure: SourceData F2.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal