Journal: bioRxiv

Article Title: Kinetics of local C3 production orchestrates neutrophil recruitment in lung injury

doi: 10.64898/2026.02.17.706326

Figure Lengend Snippet: ( A ) Schematic of infection in mouse model. Created in BioRender. Kulkarni, H. (2026) https://BioRender.com/jevlom7 ( B-C ) Assay for detecting C3 activation on lipopolysaccharide (LPS) normalized to wildtype (WT) uninfected ( B ) bronchoalveolar lavage (BAL) and ( C ) serum of WT mice infected with Pseudomonas (Pa) and euthanized at 2, 4, 8, 12 and 24 h. Results for ( D ) full-length C3 ELISA and ( E ) C3a-neoepitope ELISA in BAL post- Pa infection. ( F ) Schematic of infection in ex vivo human lung model. Modified based on Zhang et al. ( G-I ) Changes in BAL ( G ) C3, ( H ) C3b/iC3b, and ( I ) IgM levels in ex vivo human lung pre-infection (T0, 0 h) and post-infection (T4, 4h). Distribution of data is represented as scatterplots showing individual data points, and bars represent means ± SD. ****P<0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05 using ordinary one-way ANOVA after adjusting for multiple group comparisons, and Mann-Whitney test for two-group comparison. Abbreviations: CFU: colony-forming units; FACS: fluorescence-associated cell sorting (flow cytometry); PEEP: positive end-expiratory pressure; RR: respiratory rate; VT: tidal volume.

Article Snippet: For use with BAL and serum, 96-well flat-bottom ELISA plates (#3855, Thermo Fisher Scientific) were coated with a C3b capture antibody (1:50 dilution; 100 μl per well in PBS; #HM1045, rat anti-mouse C3b/iC3b/C3d/C3dg, clone 11H9; Hycult Biotech) and incubated overnight at 4°C.

Techniques: Infection, Activation Assay, Enzyme-linked Immunosorbent Assay, Ex Vivo, Modification, MANN-WHITNEY, Comparison, Fluorescence, FACS, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Elongator is required for pattern recognition receptor and type I interferon signaling in macrophages

doi: 10.1016/j.jbc.2025.110916

Figure Lengend Snippet: Differential protein expression in Elp3 −/− BMDMs relative to WT cells . Volcano plots showing differentially expressed proteins in unstimulated ( A ), 6 h LPS-treated ( B ) or 12 h LPS-treated ( C ) Elp3 −/− lysates versus lysates from WT cells. To capture a broad range of biological processes potentially influenced by loss of ELP3 an enrichment cut-off of a –log10 ( p -value) of 1.3 was considered significant. Proteins associated with IFN signalling by gene ontology biological process terms are labelled as red dots . IFN, interferon; LPS, lipopolysaccharide.

Article Snippet: ELISA plates were coated with a monoclonal rat anti-mouse IFNβ capture antibody (Santa Cruz, Cat#SC-57201) in carbonate buffer overnight at 4 °C.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Elongator is required for pattern recognition receptor and type I interferon signaling in macrophages

doi: 10.1016/j.jbc.2025.110916

Figure Lengend Snippet: Impaired LPS-IFN I signalling axis in Elp3 −/− BMDMs . A , schematic of LPS- IFN-I signalling axis. Figure created in BioRender ( B – K ) Label free quantification intensity values detected by mass spectrometry in WT and Elp3 −/− cells stimulated for 0, 6 or 12 h with LPS for peptides from transcription factor proteins IRF3 ( B ), IRF5 ( C ), IRF7 ( D ), IRF9 ( E ), RELA ( F ), NFKB1 ( G ), NFKB2 ( H ), STAT1 ( I ), STAT3 ( J ) and STAT6 ( K ). ND, not detected. Data are mean ± SD for quadruplicate (or triplicate for WT 0 h sample) measurements. ∗ p < 0.05 compared to WT, based on Mann-Whitney test. L – N , WT and Elp3 −/− cells were stimulated with LPS (100 ng/ml) for 3, 6 and 24 h. Ifnb ( L ), Irf7 ( M ) and Ifna ( N ) mRNA were assayed by qRT-PCR, expressed relative to the untreated WT control and normalized to the housekeeping gene β-actin. Data are mean ± SD of three independent experiments. Data significance was tested with a 2-way ANOVA using Šídák's multiple comparisons test. ∗ p < 0.05 and ∗∗ p < 0.0001 compared to WT. O , cells were stimulated with LPS for the indicated times and supernatants assayed by ELISA for IFN-β. Data are mean ± SD for three independent experiments, each performed on three WT or Elp3 −/− clones in triplicate. ∗∗∗ p < 0.001. LPS, lipopolysaccharide.

Article Snippet: ELISA plates were coated with a monoclonal rat anti-mouse IFNβ capture antibody (Santa Cruz, Cat#SC-57201) in carbonate buffer overnight at 4 °C.

Techniques: Quantitative Proteomics, Mass Spectrometry, MANN-WHITNEY, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Clone Assay

Journal: The Journal of Biological Chemistry

Article Title: Elongator is required for pattern recognition receptor and type I interferon signaling in macrophages

doi: 10.1016/j.jbc.2025.110916

Figure Lengend Snippet: ELP3 is required for IFN I and TYK 2-dependent signaling . A , WT and Elp3 −/− BMDMs were stimulated with 1000 U/ml IFNβ for the indicated times. Cells were harvested and immunoblotted for phosphorylated STAT1, STAT1 and β-actin. Representative of three independent experiments. B – D , WT and Elp3 −/− BMDMs were stimulated with 1000 U/ml IFNβ for the indicated times. RNA was isolated and mRNA expression of Irf7 ( B ), Isg15 ( C ), and Stat1 ( D ) were assayed by qRT-PCR, expressed relative to the untreated WT control and normalized to β-actin. E , WT and Elp3 −/− iBMDMs were stimulated with either LPS (100 ng/ml), IFNβ (1000 U/ml) or IFNγ (25 ng/ml) for 90 min. Cells were harvested and expression of the indicated proteins was assessed by immunoblot. Representative of 3 independent experiments. F , WT and Elp3 −/− iBMDMs were stimulated with either LPS (100 ng/ml), IFNβ (1000 U/ml) or IFNγ (25 ng/ml) for 3 h. Irf1 gene expression was assessed by qRT-PCR. mRNA levels are represented relative to β-actin, with each WT sample set to 100% and Elp3 −/− mRNA levels expressed as a percentage of their WT stimulated counterpart. G , WT and Elp3 −/− iBMDMs were stimulated with IL-10 (10 ng/ml) for 3 h. Socs3 mRNA was assayed by qRT-PCR, expressed relative to the untreated WT control and normalized to the housekeeping gene β-actin. H , WT and Elp3 −/− iBMDMs were stimulated with IL-4 (10 ng/ml) for 3 h. Arg1 mRNA was assayed by qRT-PCR, expressed relative to the untreated WT control and normalized to the housekeeping gene β-actin. For ( B – D and F – H ), data are mean ± SD of 3 independent experiments, each performed in triplicate. Data significance was tested with a 2-way ANOVA using Šídák's multiple comparisons test. ∗ p < 0.05 and ∗∗ p < 0.01 and ∗∗∗ p < 0.001 compared to WT. iBMDM, immortalised bone marrow–derived macrophage; LPS, lipopolysaccharide.

Article Snippet: ELISA plates were coated with a monoclonal rat anti-mouse IFNβ capture antibody (Santa Cruz, Cat#SC-57201) in carbonate buffer overnight at 4 °C.

Techniques: Isolation, Expressing, Quantitative RT-PCR, Control, Western Blot, Gene Expression, Derivative Assay