Journal: Frontiers in Immunology

Article Title: Inhaled bovine lactoferrin modulates the p47phox–MPO–NETosis axis in acute lung injury: implications for bioengineered nanomedicine in respiratory infections

doi: 10.3389/fimmu.2026.1760949

Figure Lengend Snippet: GSEA was performed on the entire list of captured proteins from all four time points, regardless of their differential expression status, using the KEGG pathway database. A Venn diagram analysis of all enriched pathways across the time points (A) identified five pathways uniquely and commonly enriched at both D1 and D3, including “NET formation”, “ECM-receptor interaction”, “Circadian entrainment”, “Hypertrophic cardiomyopathy”, “Arrhythmogenic right ventricular cardiomyopathy”, and “Retrograde endocannabinoid signaling”. Notably, “NET formation” was the only pathway that overlapped with those identified in previous analyses. The proteins enriched in this pathway and LTF are displayed in a heatmap (B, C) , showing the upregulation of proteins such as MPO, p47phox, PADI4, NCF2, and NCF4. The GSEA enrichment score plots for the “NET formation” pathway at the D1 and D3 time points are shown in (D, E) , respectively.

Article Snippet: Antibodies used in western blotting were: LTF (Proteintech, China, #31267-1-AP), MPO (Immunoway, USA, YM8618), p47phox (Proteintech, China, #28187-1-AP), p-p47phox (Immunoway, China, #YP0828) and β-actin (QuaYad, China, #QYA10734A).

Techniques: Quantitative Proteomics

Journal: Frontiers in Immunology

Article Title: Inhaled bovine lactoferrin modulates the p47phox–MPO–NETosis axis in acute lung injury: implications for bioengineered nanomedicine in respiratory infections

doi: 10.3389/fimmu.2026.1760949

Figure Lengend Snippet: Exogenous bLF attenuates LPS-induced acute lung injury and oxidative stress response at D1. Mice were divided into three groups ( n = 8 per group): CON_D1 (saline control), LPS_D1 (5 mg/kg LPS), and bLF_D1 (bLF administered via pulmonary delivery 1-2 h before LPS). (A) ELISA of lung homogenates ( n = 8) showing that LPS-induced elevations in IL-1β, IL-6, and TNF-α were reduced by bLF treatment, while the decreased anti-inflammatory cytokine IL-10 was restored and increased after bLF administration. (B) H&E staining ( n = 8) demonstrating that alveolar hemorrhage, neutrophil infiltration, and interstitial thickening in LPS_D1 lungs were alleviated in the bLF_D1 group. (C, D) Immunofluorescence staining ( n = 8) of Ly6G and MPO revealed increased fluorescence intensity following LPS stimulation, which was suppressed by bLF treatment. (E, F) qPCR analysis of lung tissue ( n = 8) indicating that LPS upregulates the mRNA expression of Ltf, Mpo, Ncf1, while bLF treatment reversed these changes. (G, H) Western blot analysis ( n = 4) confirmed that protein levels of MPO, p47phox, and p-p47phox were elevated in the LPS_D1 group and downregulated after bLF treatment. * p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant.

Article Snippet: Antibodies used in western blotting were: LTF (Proteintech, China, #31267-1-AP), MPO (Immunoway, USA, YM8618), p47phox (Proteintech, China, #28187-1-AP), p-p47phox (Immunoway, China, #YP0828) and β-actin (QuaYad, China, #QYA10734A).

Techniques: Saline, Control, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Fluorescence, Expressing, Western Blot

Journal: Biomedicines

Article Title: Disparate Roles of Oxidative Stress in Rostral Ventrolateral Medulla in Age-Dependent Susceptibility to Hypertension Induced by Systemic l -NAME Treatment in Rats

doi: 10.3390/biomedicines10092232

Figure Lengend Snippet: Effect of systemic l -NAME treatment on reactive oxygen species (ROS) levels and expression of proteins involved in ROS production and degradation. Data showing ROS levels in RVLM ( A ) and MDA levels in plasma ( B ) of young (8 weeks, open circles or squares) or adult (20 weeks, filled circles or squares) rats after i.p. infusion of saline or l -NAME (10 mg/kg/day) for 14 days. Also shown are representative gels (insets) and densitometric analysis of results from Western blot changes in protein expression of pg91 phox , p22 phox , p47 phox , and p67 phox ( C ) or SOD2 and Nrf2 ( D ), as well as enzyme activity of NADPH oxidase and total antioxidants ( E ) in RVLM 14 days after saline or l -NAME treatment. Data on protein expression are normalized to the respective saline control value, which is set to 1.0. Data are presented as mean ± SD, n = 6 per group. * p < 0.05 versus corresponding saline-treated group in unpaired Student’s t -test.

Article Snippet: Primary antisera used in this study included goat polyclonal, rabbit polyclonal or monoclonal, or mouse monoclonal antiserum against gp91 phox (1:5000; BD Biosciences, Sparks, MD, USA), p22 phox , p67 phox , and p47 phox (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), SOD2 (1:3000; Stressgen, San Deigo, CA, USA), Nrf2 (1:1000; Santa Cruz), nNOS, iNOS, and eNOS (1:1000; BD Biosciences), Noxa1 (1:1000, Santa Cruze), UCP3 (1:1000, Santa Cruz), and GAPDH (1:10,000; Merck).

Techniques: Expressing, Clinical Proteomics, Saline, Western Blot, Activity Assay, Control

Journal: Redox biology

Article Title: Clozapine suppresses NADPH oxidase activation, counteracts cytosolic H 2 O 2 , and triggers early onset mitochondrial dysfunction during adipogenesis of human liposarcoma SW872 cells.

doi: 10.1016/j.redox.2023.102915

Figure Lengend Snippet: Fig. 2. Clozapine blunts NOX-2-dependent ROS in SW872 cells. Confluent SW872 cells were induced to differentiate for 3 days with or without CLZ. (A) ROS formation detected with a flow cytometric assay using DCF. (B) Representative micrographs showing phosphorylated p47phox localization (white arrows). In these experiments, the cells were fixed and analysed for immunocy tochemical detection of phosphorylated p47phox by confocal microscopy, with all three panels having identical excitation and exposure conditions. Magnification 60X; Scale bar represents 25 μm. (C) Representative flow cytometry histograms of phosphorylated p47phox fluorescence. (D) Statistical analysis of the results obtained in (C). (E–F) Statistical analysis of flow cytometry histograms of p47phox and p67phox fluorescence. T3 CTR received DMSO alone. Data are expressed in % respect to T0 condition. Results represent the means ± SEM calculated from at least three independent determinations. *p < 0.05, **p < 0.01, as compared to T0; (**)p < 0.01, as compared to T3. ns: not significant (one-way ANOVA followed by Dunnett’s test).

Article Snippet: Anti-phosphorylated p47phox (STJ91180; St. John’s Laboratory, UK), anti-p67phox (CQA1036; Cohesion Biosciences) and anti-p47phox (SAB4502810; Merck Life Science s.r.l) were incubated for 30 min at 4 ◦C; after a washing step, anti-rabbit IgG-FITC antibody was added (sc-2012; Santa Cruz Biotech, Santa Cruz CA), as indicated in the manufacturer’s instructions.

Techniques: Flow Cytometry, Confocal Microscopy, Fluorescence

Journal:

Article Title: Cross-talk between IRAK-4 and the NADPH oxidase ¹

doi: 10.1042/BJ20061184

Figure Lengend Snippet: IRAK-4-dependent phosphorylated sites in p47phox were detected using microcapillary reversephase HPLC–nanoelectrospray and targeted ion MS/MS as described in the Experimental section. The upper panel shows a schematic representation of p47phox. The phox homology (PHOX) domain is shown in black, the SH3 domains are shown in white, and the phosphorylation domains are in grey. The lower panel shows the sequences surrounding the phosphorylated residues detected by HPLC–MS/MS. Phosphorylated residues are preceded by ‘p’. Owing to the proximity of Ser147, Thr148 and Thr150, the precise phosphorylation site could not be resolved, although a definitive phosphorylation site exists at these residues. For the complete MS/MS spectra, see Supplementary Figure S1 at http://www.BiochemJ.org/bj/403/bj4030451add.htm.

Article Snippet: Endogenous p47 phox (red) and endogenous IRAK-4 (green) were immunolabelled using a mouse monoclonal antibody (clone 0.33) and a rabbit polyclonal antibody (ProSci) respectively, as described in the Experimental section.

Techniques: Tandem Mass Spectroscopy, Phospho-proteomics

Journal: American journal of physiology. Heart and circulatory physiology

Article Title: Association of testosterone with estrogen abolishes the beneficial effects of estrogen treatment by increasing ROS generation in aorta endothelial cells.

doi: 10.1152/ajpheart.00681.2014

Figure Lengend Snippet: Fig. 6. NADPH-oxidase expression and phosphorylation in aortas of female SHRs. Shown are mRNA expression of endothelial nitric oxide synthase (eNOS; A) and NADPH-oxidase subunits p22phox (B) and p47phox (C), NOX1 (D), NOX2 (E), and NOX4 (F), normalized to the expression of -actin. G: results of immunoblots analyzed in single aortas and probed with antibodies against phosphorylated (p)-serine-345-p47phox or total (t)-p47phox, as indicated, and plotted as the ratio of p-p47phox to t-p47phox. Analysis was performed in aortas from Sham, OVX, OVXCEE, and OVXCEET rats. Data represent the mean SE from 6–8 independent experiments. *P 0.05.

Article Snippet: After blockade for 1-h blocking with 5% nonfat milk in Tris-buffered saline with 0.1% (vol/vol) Tween 20 (TBST), membranes were incubated overnight at 4°C with 1:1,000 dilution of polyclonal phospho-Ser345 p47phox antibody (Biorbyt, Cambridge, UK).

Techniques: Expressing, Phospho-proteomics, Western Blot