G3BP1 assists p38α in phosphorylating eIF6 and promotes ILC3 immune response. (A) MNK-3 cells were overexpressed with eIF6WT or mutant eIF6 (eIF6S243A and eIF67ST-A). The IL-22 and GM-CSF production was analyzed by flow cytometry. (B and C) eIF6-interactome were determined by immunoprecipitation with anti-eIF6 antibody followed by MS in ILC3s. (B) The Venn diagram shows the interactive analysis with the eIF6-interactome and TurboID-p38α-interactome. (C) The GO enrichment analysis of 35 proteins interacted with both eIF6 and p38α. (D) The p38α docking sequences in G3BP1. (E) Western blot analysis of the co-IP between p-p38α and G3BP1 or between eIF6 and G3BP in MNK-3 cells treated with or without IL-1β. (F) The binding affinity between G3BP1 and eIF6 (fixed phase) was measured by Biacore SPR assay. (G) eIF6 was incubated with p38α kinase buffer with or without G3BP1 protein in vitro. Western blot analysis of phosphorylated eIF6 following the kinase assay and Phos-tag SDS-PAGE. (H) The small intestine and colon LPLs from WT mice were pre-treated with EGCG and then stimulated with IL-1β or IL-23 for 4 h. The expression of IL-22 and GM-CSF in ILC3s (gated in eflour780−CD45lowCD90high) were analyzed by flow cytometry. (I and J) ILC3s sorted from the small intestine and colon LPLs of Rag1−/− mice were stimulated with IL-1β with or without EGCG for 18 h. The protein (I) and mRNA (J) expression levels of IL-22 and GM-CSF were examined by ELISA and qPCR. Data are representative of one of two independent experiments (A and E–J). Each dot (A and H–J) represents one biological replicate; Biacore Insight Evaluation software (GE Healthcare) is applied to calculated the KD (F). Data are mean ± SEM and statistical significance was tested by two-tailed Student’s unpaired t test (B and K), ***P < 0.001, ****P < 0.0001; ns, no significant difference. Source data are available for this figure: SourceData F7.
Figure 7.

G3BP1 assists p38α in phosphorylating eIF6 and promotes ILC3 immune response. (A) MNK-3 cells were overexpressed with eIF6WT or mutant eIF6 (eIF6S243A and eIF67ST-A). The IL-22 and GM-CSF production was analyzed by flow cytometry. (B and C) eIF6-interactome were determined by immunoprecipitation with anti-eIF6 antibody followed by MS in ILC3s. (B) The Venn diagram shows the interactive analysis with the eIF6-interactome and TurboID-p38α-interactome. (C) The GO enrichment analysis of 35 proteins interacted with both eIF6 and p38α. (D) The p38α docking sequences in G3BP1. (E) Western blot analysis of the co-IP between p-p38α and G3BP1 or between eIF6 and G3BP in MNK-3 cells treated with or without IL-1β. (F) The binding affinity between G3BP1 and eIF6 (fixed phase) was measured by Biacore SPR assay. (G) eIF6 was incubated with p38α kinase buffer with or without G3BP1 protein in vitro. Western blot analysis of phosphorylated eIF6 following the kinase assay and Phos-tag SDS-PAGE. (H) The small intestine and colon LPLs from WT mice were pre-treated with EGCG and then stimulated with IL-1β or IL-23 for 4 h. The expression of IL-22 and GM-CSF in ILC3s (gated in eflour780−CD45lowCD90high) were analyzed by flow cytometry. (I and J) ILC3s sorted from the small intestine and colon LPLs of Rag1−/− mice were stimulated with IL-1β with or without EGCG for 18 h. The protein (I) and mRNA (J) expression levels of IL-22 and GM-CSF were examined by ELISA and qPCR. Data are representative of one of two independent experiments (A and E–J). Each dot (A and H–J) represents one biological replicate; Biacore Insight Evaluation software (GE Healthcare) is applied to calculated the KD (F). Data are mean ± SEM and statistical significance was tested by two-tailed Student’s unpaired t test (B and K), ***P < 0.001, ****P < 0.0001; ns, no significant difference. Source data are available for this figure: SourceData F7.

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