DBR1 is essential for G3BP protein stability and antiviral effect in vitro. (A) Alignment of the N-terminal sequences of the DBR1 proteins of the indicated species. (B) WB detection with the indicated antibodies in MEFs. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (C) WB detection with the indicated antibodies in BMDMs. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (D) Fluorescence detection of the indicated markers in MEF cells. Scale bars, 20 µm. Data shown are representative of three independent experiments. (E) WT and Dbr1Y17H/Y17H MEFs were infected with VSV (MOI: 0.1) or HSV-1 (MOI: 0.5) for 24 h, stimulated with polyI:C (2 µg/ml) for 5 h or incubated at 43°C for 40 min; fluorescence imaging was then performed for the indicated markers. Scale bars, 20 µm. Data shown are representative of three independent experiments. (F) Detection of fluorescent immunostaining for the indicated markers in WT and Dbr1Y17H/Y17H mouse brain sections. Scale bars, 50 µm. Data shown are representative of three independent experiments. (G) Fluorescence imaging of WT and Dbr1Y17H/Y17H MEFs infected with HSV-1-GFP (MOI: 0.5) for various time points. Scale bars, 10 0 µm. Data shown are representative of five independent experiments. (H and I) WT and Dbr1Y17H/Y17H MEFs were infected with VSV at a MOI of 0.1 (H) or HSV-1 at a MOI of 0.5 (I) for 36 h. RT-qPCR was then performed to detect the expression of the indicated viral genes. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ****, P < 0.0001. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. (J and K) Similar to H and I, except that WB was performed with the indicated antibodies after infection with VSV (J) or HSV-1 (K). Tubulin (K) or GAPDH (J) was used as a loading control. Data shown are representative of three independent experiments. (L–N) WT, Dbr1Y17H/+, and Dbr1Y17H/Y17H MEFs were infected with VSV (MOI: 0.1) for 24 h, and RT-qPCR was then performed to detect expression of the indicated genes. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. NS, not significant, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Source data are available for this figure: SourceData F7.
Figure 7.

DBR1 is essential for G3BP protein stability and antiviral effect in vitro. (A) Alignment of the N-terminal sequences of the DBR1 proteins of the indicated species. (B) WB detection with the indicated antibodies in MEFs. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (C) WB detection with the indicated antibodies in BMDMs. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (D) Fluorescence detection of the indicated markers in MEF cells. Scale bars, 20 µm. Data shown are representative of three independent experiments. (E) WT and Dbr1Y17H/Y17H MEFs were infected with VSV (MOI: 0.1) or HSV-1 (MOI: 0.5) for 24 h, stimulated with polyI:C (2 µg/ml) for 5 h or incubated at 43°C for 40 min; fluorescence imaging was then performed for the indicated markers. Scale bars, 20 µm. Data shown are representative of three independent experiments. (F) Detection of fluorescent immunostaining for the indicated markers in WT and Dbr1Y17H/Y17H mouse brain sections. Scale bars, 50 µm. Data shown are representative of three independent experiments. (G) Fluorescence imaging of WT and Dbr1Y17H/Y17H MEFs infected with HSV-1-GFP (MOI: 0.5) for various time points. Scale bars, 10 0 µm. Data shown are representative of five independent experiments. (H and I) WT and Dbr1Y17H/Y17H MEFs were infected with VSV at a MOI of 0.1 (H) or HSV-1 at a MOI of 0.5 (I) for 36 h. RT-qPCR was then performed to detect the expression of the indicated viral genes. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ****, P < 0.0001. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. (J and K) Similar to H and I, except that WB was performed with the indicated antibodies after infection with VSV (J) or HSV-1 (K). Tubulin (K) or GAPDH (J) was used as a loading control. Data shown are representative of three independent experiments. (L–N) WT, Dbr1Y17H/+, and Dbr1Y17H/Y17H MEFs were infected with VSV (MOI: 0.1) for 24 h, and RT-qPCR was then performed to detect expression of the indicated genes. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. NS, not significant, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Source data are available for this figure: SourceData F7.

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