Journal: bioRxiv

Article Title: Herpes simplex virus infection promotes ALS pathology through ICP0-mediated PML body disruption

doi: 10.64898/2026.03.27.714707

Figure Lengend Snippet: a , Schematic overview of the HSV-1 ICP0 gene with mutations to disrupt either RNF8 binding site (T67A), E3 ligase function (C116G/C156A), Cyclin D3 binding site (D199A) or CoREST interaction (D671A/E673A). b , Representative immunofluorescence images of H4 cells co-transfected with TDP-43venus (green) and either vector, wild-type (WT) ICP0 or indicated ICP0 mutant. Cells were fixed 24 h post-transfection and stained for PML (white), ICP0 (red) and DAPI (blue). Scale bar, 10 µm. c , Quantification of nuclear TDP-43venus foci area (µm 2 ) (top) and PML foci count (bottom) of co-transfected cells in (b) using Foci Analyzer (ImageJ). Lines represent mean±SEM, dots represent individual cells (n = 37-74) of three independent biological replicates. *p<0.05, ***p<0.001 Kruskal-Wallis test followed by Dunn’s multiple comparisons test. d , Scatter plot of total nuclear TDP-43venus foci size (y-axis) against the nuclear PML dot count (x-axis). Each dot represents one corresponding measurement from (c). Blue dots represent cells overexpressing ICP0 WT, black dots represent cells transfected with vector. For association testing, all events were taken together in Spearman’s rank correlation test (r = –0.54, ***p<0.001, n = 118). e , Representative immunofluorescence images of H4 cells transduced with lentiviruses encoding TDP-43venus (green). After 3 days, cells were infected with HSV-1 and 1 h post-infection treated with proteasome inhibitor MG132 or DMSO, respectively. Cells were fixed 17 h post-infection and stained for PML (white), ICP27 (red) and DAPI (blue). Scale bar, 10 µm. f , Quantification of PML foci (top) and total nuclear TDP-43venus foci area (µm 2 ) (bottom) in (e). Lines represent mean±SEM. Dots represent individual cells, n = 58-362, three independent biological replicates, ***p<0.001 Kruskal-Wallis test followed by Dunn’s multiple comparisons test. g , Representative immunoblots showing SUMOylation status of FLAG-tagged TDP-43 purified by anti-FLAG immunoprecipitation (IP) from whole cell lysates (WCL) of HEK293T cells at 40 h post-transfection. Cells were co-transfected with HSV-1 ICP0 and/or stimulated with arsenite (500 µM, 1 h), as indicated. IP and WCL was analyzed by immunoblotting. Blots were stained with anti-SUMO2, anti-FLAG, anti-HSV-1 ICP0 and anti-GAPDH. h , Quantification of the full SUMO-2 lane in the TDP-43 IP of ( g ) normalized to the vector control. n = 3, **p<0.01 paired t test.

Article Snippet: Monoclonal mouse anti-FLAG-tag antibody, M2 (1:5,000), Sigma-Aldrich, F1804), anti-GAPDH (BioLegend, 607902; 1:1,000), Polyclonal rabbit anti-TDP-43 antibody (proteintech, 10782-2-AP, 1:2,000), mouse anti-lamin B (proteintech, 66095-1, 1:1,000), mouse anti-HSV-1 ICP0 (abcam, AB6513, 1:7,500), anti-SUMO2 (novus biologicals, NBP1-77163, 1:1,000) Secondary Antibodies: anti-rabbit IgG HRP (Promega, W401B, 1:10,000), anti-mouse IgG HRP (Promega, W402B, 1:10,000), Veriblot HRP (abcam, ab131366, 1:1,000)

Techniques: Binding Assay, Immunofluorescence, Transfection, Plasmid Preparation, Mutagenesis, Staining, Transduction, Infection, Western Blot, Purification, Immunoprecipitation, Control

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a Immunofluorescence images and quantification (n = 20) of SUMO1 (green) and SUMO2/3 (red) conjugation in RAW264.7 cells with or without STM 14028S infection (4 hpi). Scale bar, 10 µm. b Core SUMO cycle enzymes and their corresponding primary genes (blue). c, d Transcriptomic and proteomic analyses of SUMO cycle enzyme expression in RAW264.7 cells infected with STM 14028S vs uninfected controls (4 hpi; n = 3). e UBC9 mRNA expression in RAW264.7 cells during STM 14028S infection (0-6 hpi; n = 3). f Immunoblot analysis and quantification of UBC9 protein levels in STM 14028S-infected RAW264.7 cells (0-6 hpi; n = 3). *P < 0.05; ***P < 0.001; ns, not significant.

Article Snippet: Unique primary antibodies used in this study included UBC9 (CST, #4786), SUMO1 (Proteintech, 67557-1-lg), SUMO2/3 (Proteintech, 67154-1-lg), arginine-GlcNAcylation antibody (Abcam, EPR18251), Myd88 (Proteintech, 67969-1-lg), HSPA8 (Proteintech, 10654-1-AP), PDCD4 (Proteintech, 84162-3-RR).

Techniques: Immunofluorescence, Conjugation Assay, Infection, Expressing, Western Blot

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a Immunofluorescence images and quantification (n = 20) of SUMO1 (green) and SUMO2/3 (red) conjugation in RAW264.7 cells infected with STM 14028S over a 0-6 hrs time course. Scale bar, 10 µm. b Immunofluorescence images and quantification (n = 20) of SUMO1 (green) and SUMO2/3 (red) conjugation in RAW264.7 cells infected with STM WT or Δ ssaV or left uninfected (4 hpi). Scale bar, 10 µm. c Schematic of the high-content screening (HCS) workflow used to identify T3SS-2 effector(s) required for subversion of host SUMOylation. d SUMO2/3 suppression rates in RAW264.7 cells infected with STM WT or T3SS-2 effector knockout strains (4 hpi). For each sample, fluorescence intensity (FI) was measured across three fields (20 cells per field) to calculate the mean FI (MFI). Suppression rate = (MFI_uninfected - FI_test) / (MFI_uninfected - MFI_WT). e Representative immunofluorescence images of SUMO2/3 conjugation in RAW264.7 cells infected with STM WT, Δ ssaV , Δ sseK1 , or left uninfected (4 hpi). f Immunoblot analysis of SUMO2/3 conjugates in RAW264.7 cells infected with STM WT or Δ sseK1 or left uninfected (4 hpi). *P < 0.05; ***P < 0.001; ns, not significant.

Article Snippet: Unique primary antibodies used in this study included UBC9 (CST, #4786), SUMO1 (Proteintech, 67557-1-lg), SUMO2/3 (Proteintech, 67154-1-lg), arginine-GlcNAcylation antibody (Abcam, EPR18251), Myd88 (Proteintech, 67969-1-lg), HSPA8 (Proteintech, 10654-1-AP), PDCD4 (Proteintech, 84162-3-RR).

Techniques: Immunofluorescence, Conjugation Assay, Infection, High Content Screening, Knock-Out, Fluorescence, Western Blot

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a Immunoblot analysis of UBC9 Arg-GlcNAcylation mediated by SseK1, SseK2, or SseK3. b AlphaFold-predicted structures of SseK1 (aa 29-336, red), SseK2 (aa 29-348, yellow), and SseK3 (aa 29-335, green), shown with structural alignment using PyMOL. c AlphaFold-modeled structure of the SseK1-UBC9 complex. The enlarged view of the lid-domain region is boxed in black. Residues in the SseK1 lid domain forming hydrogen bonds with UBC9 are shown in stick representation. d Sequence alignment of SseK1, SseK2, and SseK3 generated using ESPript 3.0. e Immunoblot analysis of UBC9 Arg-GlcNAcylation mediated by SseK1, SseK1 A332_Q336del, SseK3, and SseK3 R332delinsARHVQ. f Immunoblot analysis of SUMO2/3 conjugation in RAW264.7 cells infected with STM Δ sseK1 , STM Δ sseK1 complemented with SseK1, or STM Δ sseK1 complemented with either SseK1 D223_D225delinsAAA or SseK1 A332_Q336del (4 hpi). g Phylogenetic analysis of Salmonella Typhimurium SseK1 homologs. Protein sequences homologous to SseK1 (UniProt accession: A0A0H3NK84) were identified using BLASTP analysis against the UniProtKB reference proteomes and Swiss-Prot databases. Sequences with an E-value < 0.05 were selected for phylogenetic analysis. The phylogenetic tree was constructed and visualized using the Interactive Tree of Life (iTOL) online tool.

Article Snippet: Unique primary antibodies used in this study included UBC9 (CST, #4786), SUMO1 (Proteintech, 67557-1-lg), SUMO2/3 (Proteintech, 67154-1-lg), arginine-GlcNAcylation antibody (Abcam, EPR18251), Myd88 (Proteintech, 67969-1-lg), HSPA8 (Proteintech, 10654-1-AP), PDCD4 (Proteintech, 84162-3-RR).

Techniques: Western Blot, Sequencing, Generated, Conjugation Assay, Infection, Construct

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a Protein SUMOylation sites showing a >3-fold increase in SUMOylation intensity in STM Δ sseK1 -infected cells relative to WT. SUMOylation intensity was normalized to the corresponding protein abundance measured in proteomic datasets from STM Δ sseK1 - or WT-infected RAW264.7 cells. b GO and KEGG pathway enrichment analysis of proteins with SUMOylation intensity ratio (Δ sseK1 /WT) > 3. c, d IP analysis of Myd88 and Hspa8 SUMO2/3 modification in RAW264.7 cells infected with STM WT or Δ sseK1 or left uninfected (4 hpi). e Immunoblot analysis of SUMO2/3 modification in RAW-SseK1-OE and RAW-GFP-Ctrl cells. f Nine-quadrant plot showing protein abundance changes in two proteomic comparisons (RAW-SseK1-OE vs RAW-GFP-Ctrl and STM WT-infected vs Δ sseK1 -infected cells). Highlighted proteins are downregulated in both datasets. Fold-change values within dashed lines indicate <|1.3|. g Immunoblot analysis of PDCD4 expression in RAW-SseK1-OE and RAW-GFP-Ctrl cells. h Immunoblot analysis of PDCD4 and SUMO2/3 levels in RAW264.7 cells treated with increasing concentrations of the SUMOylation inhibitor 2-D08. ***P < 0.001.

Article Snippet: Unique primary antibodies used in this study included UBC9 (CST, #4786), SUMO1 (Proteintech, 67557-1-lg), SUMO2/3 (Proteintech, 67154-1-lg), arginine-GlcNAcylation antibody (Abcam, EPR18251), Myd88 (Proteintech, 67969-1-lg), HSPA8 (Proteintech, 10654-1-AP), PDCD4 (Proteintech, 84162-3-RR).

Techniques: Infection, Quantitative Proteomics, Modification, Western Blot, Expressing

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a Intracellular survival of STM WT and Δ sseK1 strains in RAW264.7 cells (1-6 hpi). n = 3. b Schematic of the experimental workflow for 2-D08 treatment and Salmonella infection of RAW264.7 cells. c Intracellular survival of STM WT and Δ sseK1 strains in RAW264.7 cells with or without 2-D08 pretreatment (4 hpi). d Schematic of the experimental workflow for 2-D08 administration and STM challenge in C57BL/6 mice. e Immunoblot analysis of SUMO2/3 modification in liver, spleen, and intestine tissues from C57BL/6 mice challenged with STM WT or Δ sseK1 , with or without 2-D08 pretreatment. f Bacterial load (CFU per gram of liver or spleen) in C57BL/6 mice infected with STM WT or Δ sseK1 . n = 4. g Survival curves of C57BL/6 mice challenged with STM WT or Δ sseK1 , with or without 2-D08 pretreatment. n = 6. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Article Snippet: Unique primary antibodies used in this study included UBC9 (CST, #4786), SUMO1 (Proteintech, 67557-1-lg), SUMO2/3 (Proteintech, 67154-1-lg), arginine-GlcNAcylation antibody (Abcam, EPR18251), Myd88 (Proteintech, 67969-1-lg), HSPA8 (Proteintech, 10654-1-AP), PDCD4 (Proteintech, 84162-3-RR).

Techniques: Infection, Western Blot, Modification