Journal: Nature Communications

Article Title: Shigella flexneri evades septin-mediated cell-autonomous immunity via protein ADP-riboxanation

doi: 10.1038/s41467-026-68425-0

Figure Lengend Snippet: a Coomassie blue-stained purified 3×FLAG-SEPT9 from 293T cells co-transfected with GFP-OspC3-WT or GFP-OspC3-EH/AA prior to MS analyses. Right: ClustalW2 multiple sequence alignment of SEPT3 subgroup members. Key salt-bridge residues at the NC-interface are marked with yellow circles. ADP-riboxanation sites are highlighted in red; asterisks indicate conserved residues. The six arginine residues labeled in black were identified after the initial four sites were mutated to lysine (4RK). b Extracted ion chromatograms of an unmodified peptide, an Arg561-containing peptide and its ADP-riboxanated form from immunoprecipitated SEPT9 in ( a ). c Tandem mass spectrum of the ADP-riboxanated Arg561-containing peptide acquired under collision-induced dissociation (CID). d , e Validation of MS-identified sites by site-directed mutagenesis. 293T cells co-transfected with GFP-OspC3 and FLAG-SEPT9 (WT or variants) were analyzed by IP and immunoblotting. 10RK refers to substitution of all ten modified arginine residues to lysine. f Prokaryotic co-expression. GST-SEPT9 was expressed in E. coli alone or with calmodulin (CaM) and OspC3 as indicated. Purified GST-SEPT9 was blotted to detect ADP-riboxanation. g In vitro reconstitution. Purified recombinant proteins were incubated (37 °C, 1 h) and analyzed by Coomassie staining and Western blotting. The effects of NAD + , CaM, and calcium ions were tested. h ADP-riboxanation during infection. HeLa cells transfected with FLAG-SEPT2, SEPT6, SEPT7, or SEPT9 were infected with S. flexneri (MOI = 50, 2 h). Anti-FLAG immunoprecipitates were analyzed by immunoblotting. i OspC-dependent modification of Arg561. HeLa cells expressing FLAG-SEPT9 (WT or R561 mutants) were infected with indicated S. flexneri strains (MOI = 50, 2 h). Modification was assessed by anti-FLAG IP and immunoblotting. UI, uninfected. j Endogenous SEPT9 modification. Analyzed as in ( i ), but immunoprecipitated with an anti-SEPT9 antibody. For ( a , d – j ), experiments were repeated three times with similar results.

Article Snippet: The clarified lysates were incubated with pre-washed anti-FLAG M2 affinity beads (Sigma-Aldrich, A2220) at 4 °C with gentle rotation for 4 h. The beads were washed four times with the lysis buffer, and bound proteins were eluted with FLAG peptides (MedChemExpress, HY-P0319A) and run on SDS-PAGE followed by either Western blot or LC-MS analyses.

Techniques: Staining, Purification, Transfection, Sequencing, Labeling, Immunoprecipitation, Biomarker Discovery, Mutagenesis, Western Blot, Modification, Expressing, In Vitro, Recombinant, Incubation, Infection

Journal: Nature Communications

Article Title: Shigella flexneri evades septin-mediated cell-autonomous immunity via protein ADP-riboxanation

doi: 10.1038/s41467-026-68425-0

Figure Lengend Snippet: a Quantitative proteomic analysis of the SEPT9 interactome upon ADP-riboxanation. 293T cells co-expressing 3×FLAG-SEPT9 with GFP-OspC3-WT or -EH/AA were analyzed by anti-FLAG IP and quantitative MS. Volcano plot shows fold changes (x-axis) vs. statistical significance (y-axis) of interacting proteins. Data represent four biological replicates. Statistical analysis: two-sided paired moderated t -test (limma). b , c Impaired septin subunit interactions. 293T cells co-transfected with EGFP-OspC3 (WT or EH/AA), FLAG-SEPT9, and HA-SEPT2 or HA-SEPT6 were analyzed by anti-FLAG IP and immunoblotting. Quantification of interaction strength is shown below blots. d Network analysis. SEPT9-associated proteins are grouped into functional clusters. Nodes are color-coded by interaction strength (log2 fold changes). e Interaction with endogenous septins. 293T cells co-transfected with EGFP-OspC3 (WT or EH/AA) and FLAG-SEPT9 (WT, 4RK, or 10RK) were analyzed by anti-FLAG IP and immunoblotting for endogenous SEPT2, SEPT6, and SEPT7. Quantification is shown below blots. f In vitro pull-down assay. Recombinant GST-Strep-tag II-SEPT9 (WT, ADP-riboxanated, 4RK, or 10RK) and GST-SEPT7 individually purified from E. coli were incubated together. Strep-Tactin pull-down samples were analyzed by immunoblotting. Quantification is shown below blots. For ( b , c , e , f ), experiments were repeated three times with similar results.

Article Snippet: The clarified lysates were incubated with pre-washed anti-FLAG M2 affinity beads (Sigma-Aldrich, A2220) at 4 °C with gentle rotation for 4 h. The beads were washed four times with the lysis buffer, and bound proteins were eluted with FLAG peptides (MedChemExpress, HY-P0319A) and run on SDS-PAGE followed by either Western blot or LC-MS analyses.

Techniques: Expressing, Transfection, Western Blot, Functional Assay, In Vitro, Pull Down Assay, Recombinant, Strep-tag, Purification, Incubation