Journal: bioRxiv

Article Title: Schlafen-11 and -9 are innate immune sensors for intracellular single-stranded DNA

doi: 10.1101/2024.02.24.581893

Figure Lengend Snippet: ( A ) Diagram of the luminous ODN immunoprecipitation (ODIP) and biotin-ODN pulldown assays. Cells were transfected with biotinylated ODN and lysed 6∼12 hours later. For ODIP, protein A/G-coated beads attached to a primary antibody were used to immunoprecipitate the protein and its bound ODNs from the cell lysate. The immunoprecipitated biotin-ODNs were then quantified using streptavidin (SA)- conjugated HRP to detect the enhanced chemiluminescence (ECL) substrate. For biotin-ODN pulldown assays, SA-coupled beads were used to pull down the biotin-ODN and the bound protein from the cell lysate. The captured proteins were analysed via immunoblotting. ( B to E ) The binding of SLFN11 to the indicated ODNs was analysed by ODIP [(B) and (D)] and biotin-ODN pulldown assays [(C) and (E)]. Biotinylated ODN60 or the indicated ODN60 mutants [(B) and (C)], or the indicated CGT ODNs and control ODNs [(D) and (E)] were transfected into HEK293 cells (C to E) or SLFN11 -/- HEK293 cells with re-expressed His-tag SLFN11 (B). Mock, transfection regent. ( F to J ) Full-length SLFN11 and the indicated truncated forms of SLFN11 (F) were re-expressed in SLFN11 -/- HEK293 cells, which were subsequently transfected with biotinylated ODN60. The binding activity of truncated SLFN11 with ODN60 was analysed by ODIP (G), and the ability of the truncated constructs of SLFN11 to rescue ODN-induced pathway activation was analysed by immunoblotting (J). ( H and I ) BioLayer Interferometry (BLI) assays of SLFN11 binding activity with CGT ODNs and control ODNs (H) and ODN-H (I). GST-tagged (H) and His-tagged (I) C-terminal SLFN11 (residues 349-901) constructs were expressed and purified from E. coli BL21 (DE3) cells and Sf9 cells, respectively. Sensorgrams reflecting the binding to ODN-H with gradient concentrations of His-tagged C-terminal SLFN11 and the calculated dissociation constants (K D ) were calculated (I). All data shown are representative of three independent experiments.

Article Snippet: To measure the direct binding between GST-c-SLFN11 (residues 349-901) and CpG ODNs, GST-c-SLFN11 was immobilized onto Anti-GST Biosensor tips (Sartorius) in BLI buffer by dipping the biosensors into wells containing GST-c-SLFN11 for 300 s. After obtaining the baseline reading, the biosensors were next transferred to wells containing the indicated biotinylated ODNs to monitor the association of ODNs with immobilized GST-c-SLFN11 for 600 s. Finally, the biosensors were transferred to wells containing BLI buffer to monitor the dissociation of ODNs from GST-c-SLFN11 protein for 600 s. To determine the kinetics of the interactions of ODN-H with His-c-SLFN11, biotinylated ODN-H was immobilized onto SA biosensor tips (Sartorius), and His-c-SLFN11 was added into wells for the association step.

Techniques: Immunoprecipitation, Transfection, Western Blot, Binding Assay, Activity Assay, Construct, Activation Assay, Purification

Journal: bioRxiv

Article Title: Schlafen-11 and -9 are innate immune sensors for intracellular single-stranded DNA

doi: 10.1101/2024.02.24.581893

Figure Lengend Snippet: ( A and B ) The binding activity of endogenous and re-expressed SLFN11 for ODN60 and ODN60rc was determined by ODIP after transfection of the indicated biotinylated ODNs into WT (A), SLFN11-deficient and SLFN11-restored (B) HEK293 cells. ( C ) The binding activity of ODN60rc or ODN60rc containing a CGT motif for SLFN11 were examined by biotin pull-down assay in HEK293 cells. ( D to G ) In vitro assays of the ODN-binding activity of purified SLFN11. GST-tagged and His-tagged SLFN11 C-terminal fragments (residues 349-901) were expressed and purified from E. coli BL21 (DE3) cells and Sf9 cells, respectively, and stained with Coomassie blue (D), Binding between the C-terminal fragment of SLFN11 and the indicated FAM-labeled ODNs was examined by electrophoretic mobility shift assay (EMSA) (E) and fluorescence ODN GST pulldown assay (F). The dissociation constant (K D ) between GST-c- SLFN11 and ODN-H was measured using a BLI assay (G). ( H to J ) Restored expression of the indicated subgroup III SLFNs from humans (H) and mice (I) with an N terminal Flag tag in SLFN11 -/- HEK293 cells. These cells were then transfected with biotinylated ODN60. The binding activity of SLFN proteins was determined by ODIP, and the ability of the indicated subgroup III SLFNs to restore the response to ODN60 was determined by immunoblotting (J). All data shown are representative of three independent experiments.

Article Snippet: To measure the direct binding between GST-c-SLFN11 (residues 349-901) and CpG ODNs, GST-c-SLFN11 was immobilized onto Anti-GST Biosensor tips (Sartorius) in BLI buffer by dipping the biosensors into wells containing GST-c-SLFN11 for 300 s. After obtaining the baseline reading, the biosensors were next transferred to wells containing the indicated biotinylated ODNs to monitor the association of ODNs with immobilized GST-c-SLFN11 for 600 s. Finally, the biosensors were transferred to wells containing BLI buffer to monitor the dissociation of ODNs from GST-c-SLFN11 protein for 600 s. To determine the kinetics of the interactions of ODN-H with His-c-SLFN11, biotinylated ODN-H was immobilized onto SA biosensor tips (Sartorius), and His-c-SLFN11 was added into wells for the association step.

Techniques: Binding Assay, Activity Assay, Transfection, Pull Down Assay, In Vitro, Purification, Staining, Labeling, Electrophoretic Mobility Shift Assay, Fluorescence, GST Pulldown Assay, Expressing, FLAG-tag, Western Blot