DBR1 licenses G3BP proteins to form SGs. (A) WB with indicated antibodies after immunoprecipitation with an anti-FLAG antibody on lysates of HEK239T cells in an overexpression system. Data shown are representative of three independent experiments. (B) BJ cells were infected with VSV at an MOI of 0.1, or HSV-1 at an MOI of 0.5 for 12 h; immunofluorescence detection was then performed for PKR, G3BP2, and G3BP1-mNeonGreen. Scale bars, 20 µm. Data shown are representative of three independent experiments. (C and D) HEK293T G3BP2-mNeonGreen/DBR1-mScarlet cells were imaged (C) and the intracellular G3BP2- mNeonGreen puncta were quantified (D). Scale bars, 50 µm. Data shown are representative of three independent experiments. 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. NS, not significant, ****, P < 0.0001. (E) Fluorescence images of HEK293T G3BP-mNeonGreen and HEK293T shDBR1/G3BP-mNeonGreen cells. Scale bars, 50 µm. Data shown are representative of three independent experiments. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. **, P < 0.01. (F and G) THP-1 cells (D) and cells from patients (E) and controls were analyzed by WB with the indicated antibodies. VCL was used as a loading control. Data shown are representative of three independent experiments. (H) G3BP1−/− HeLa cells and control cells were treated with 500 µM SA, and WB was then performed with the indicated antibodies. VCL was used as a loading control. Data shown are representative of three independent experiments. (I) G3BP DKO U2OS cells and control cells were stimulated with polyI:C (2 µg/ml) for 4 h, and WB was then performed with the indicated antibodies. VCL was used as a loading control. Data shown are representative of three independent experiments. (J) G3BP DKO HEK293T cells and control cells were stimulated with polyI:C (1 µg/ml) for various time points, and WB was then performed with the indicated antibodies. VCL was used as a loading control. Data shown are representative of three independent experiments. (K) HEK293T cells with overexpression of the indicated proteins were infected with VSV at an MOI of 0.1, and WB was then performed with the indicated antibodies, followed by the p-PKR/PKR WB gray scale analysis of three independent experiments. GAPDH was used as a loading control. Graphs depict points represent biological replicates. Data shown are representative of three independent experiments. Source data are available for this figure: SourceData FS3.
Figure S3.

DBR1 licenses G3BP proteins to form SGs. (A) WB with indicated antibodies after immunoprecipitation with an anti-FLAG antibody on lysates of HEK239T cells in an overexpression system. Data shown are representative of three independent experiments. (B) BJ cells were infected with VSV at an MOI of 0.1, or HSV-1 at an MOI of 0.5 for 12 h; immunofluorescence detection was then performed for PKR, G3BP2, and G3BP1-mNeonGreen. Scale bars, 20 µm. Data shown are representative of three independent experiments. (C and D) HEK293T G3BP2-mNeonGreen/DBR1-mScarlet cells were imaged (C) and the intracellular G3BP2- mNeonGreen puncta were quantified (D). Scale bars, 50 µm. Data shown are representative of three independent experiments. 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. NS, not significant, ****, P < 0.0001. (E) Fluorescence images of HEK293T G3BP-mNeonGreen and HEK293T shDBR1/G3BP-mNeonGreen cells. Scale bars, 50 µm. Data shown are representative of three independent experiments. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. **, P < 0.01. (F and G) THP-1 cells (D) and cells from patients (E) and controls were analyzed by WB with the indicated antibodies. VCL was used as a loading control. Data shown are representative of three independent experiments. (H) G3BP1−/− HeLa cells and control cells were treated with 500 µM SA, and WB was then performed with the indicated antibodies. VCL was used as a loading control. Data shown are representative of three independent experiments. (I) G3BP DKO U2OS cells and control cells were stimulated with polyI:C (2 µg/ml) for 4 h, and WB was then performed with the indicated antibodies. VCL was used as a loading control. Data shown are representative of three independent experiments. (J) G3BP DKO HEK293T cells and control cells were stimulated with polyI:C (1 µg/ml) for various time points, and WB was then performed with the indicated antibodies. VCL was used as a loading control. Data shown are representative of three independent experiments. (K) HEK293T cells with overexpression of the indicated proteins were infected with VSV at an MOI of 0.1, and WB was then performed with the indicated antibodies, followed by the p-PKR/PKR WB gray scale analysis of three independent experiments. GAPDH was used as a loading control. Graphs depict points represent biological replicates. Data shown are representative of three independent experiments. Source data are available for this figure: SourceData FS3.

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