Journal: Italian Journal of Animal Science

Article Title: Protective effect of chitosan oligosaccharide against oxidative damage of peripheral blood mononuclear cells in dairy cows induced by diethylenetriamine/nitric oxide via NF-κB signalling pathway

doi: 10.1080/1828051x.2020.1772131

Figure Lengend Snippet: Figure 2. Effects of chitosan oligosaccharideon on the phosphorylation level of NF-kB pathway, the protein expression level of nitric oxide synthase (iNOS) and interleukin-1b (IL-1b). Expressions of IL-1b(A); phosphorylated IjB kinase b (P-IKKb) (B); phosphorylated inhibitor of nuclear factor kappa-Ba (P-IjBa) (C); phosphorylated nuclear factor kappa-Bp65(P-NF-jBp65) (D); iNOS (E), protein levels were detected by western blotting and normalised to beta-actin (b-Actin) levels. CTR¼ control treatment, without chitosan oligosacchar- ides addition; COS40, COS80, COS160, and COS320¼ treated with 40, 80, 160, and 320lg/mL chitosan oligosaccharides respectively.

Article Snippet: The membranes were incubated in the following primary antibodies: primary polyclonal rabbit anti-iNOS antibody (NBP1-97471, Novus Biological, USA), rabbit anti-IL-1b (Cell Signalling Technology, Boston, MA), anti-IKKb, monoclonal rat anti-IjBa, monoclonal rabbit anti-NF-jBp65 antibody(NF-jB pathway Sampler Kit 9963,CST, MA), anti-phospho-IKKb, anti-phospho-IjBa, and anti-phospho-NF-jBp65 (NF-jB pathway Sampler Kit 9963, CST, MA).

Techniques: Phospho-proteomics, Expressing, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: ATF3 Stimulates IL-17A by Regulating Intracellular Ca 2+ /ROS-Dependent IL-1β Activation During Streptococcus pneumoniae Infection

doi: 10.3389/fimmu.2018.01954

Figure Lengend Snippet: ATF3 suppresses ROS generation and iNOS expression. (A) Fold change in ROS levels at 0, 2, 4, and 6 hpi compared to that in non-infected WT cells. (B) iNOS expression and (C) IHC (anti-iNOS) at 0 and 6 hpi ( n = 4). * P < 0.05, ** P < 0.01, two-way analysis of variance.

Article Snippet: The membranes were blocked with skim milk for 2 h, and then probed overnight with anti-GBP5 (gtx31537, Gentex, Zeeland, MI, USA), anti-iNOS (NB300-605, Novus International, St. Louis, MO, USA), anti-IL-1β (AF-401-SP, R&D Systems, Minneapolis, MN, USA), or anti-β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Infection

Journal: Frontiers in Immunology

Article Title: ATF3 Stimulates IL-17A by Regulating Intracellular Ca 2+ /ROS-Dependent IL-1β Activation During Streptococcus pneumoniae Infection

doi: 10.3389/fimmu.2018.01954

Figure Lengend Snippet: ATF3 plays an important role in IL-17A production in response to S. pneumoniae infection. Lung macrophages rapidly phagocytose invading S. pneumoniae during infection, resulting in ROS production, ER stress, and ATF3 activation. ATF3 then inhibits ROS-induced iNOS expression to promote NLRP3 inflammasome and GBP5 activation, triggering IL-1β secretion and the subsequent activation of γδ T cells in the lung .

Article Snippet: The membranes were blocked with skim milk for 2 h, and then probed overnight with anti-GBP5 (gtx31537, Gentex, Zeeland, MI, USA), anti-iNOS (NB300-605, Novus International, St. Louis, MO, USA), anti-IL-1β (AF-401-SP, R&D Systems, Minneapolis, MN, USA), or anti-β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Infection, Activation Assay, Expressing

Journal: Acta Pharmacologica Sinica

Article Title: Efficacy of subantimicrobial-dose doxycycline against nitrosative stress in chronic periodontitis

doi: 10.1038/aps.2012.129

Figure Lengend Snippet: Photomicrographs of representative iNOS-positive and 3-nitrotyrosine-positive stains in gingival tissue from patients with chronic periodontitis (immunohistochemical staining): (A) countless iNOS+ PMNs and monocytes (scale bar, 50 μm); (B) decreased iNOS+ cells in the SRP+placebo group (scale bar, 50 μm); (C) sparse iNOS+ cells in SRP+SDD group (scale bar, 50 μm); (D) countless 3NT+ PMNs and monocytes (scale bar, 100 μm); (E) decreased 3NT+ cells in the SRP+placebo group (scale bar, 50 μm); (F) sparse 3NT+ cells in the SRP+SDD group (scale bar, 50 μm). This figure is representative of 3 measurements (×400). iNOS+=induced nitric oxide synthase positive cells; 3NT+=3-nitrotyrosine positive cells; SRP=scaling and root planing; SDD=subantimicrobial dose of doxycycline.

Article Snippet: The following primary monoclonal antibodies were used: anti iNOS (R&D Systems, RD-MAB9502, MN, USA), anti 3-nitrotyrosine (3NT) (Novus Europe, KA0445, ABNOVA, Cambridge, UK).

Techniques: Immunohistochemical staining, Staining

Journal: Acta Pharmacologica Sinica

Article Title: Efficacy of subantimicrobial-dose doxycycline against nitrosative stress in chronic periodontitis

doi: 10.1038/aps.2012.129

Figure Lengend Snippet: Periodontal immunohystochemical and serum nitrosative stress parameters of the patients.

Article Snippet: The following primary monoclonal antibodies were used: anti iNOS (R&D Systems, RD-MAB9502, MN, USA), anti 3-nitrotyrosine (3NT) (Novus Europe, KA0445, ABNOVA, Cambridge, UK).

Techniques:

Journal: Scientific Reports

Article Title: High-intensity interval training remodels perineuronal nets in the medial prefrontal cortex to drive microglial polarization and alleviate osteoarthritis pain

doi: 10.1038/s41598-026-40823-w

Figure Lengend Snippet: HIIT reduced PNN accumulation and promoted microglial M2 polarization in the mPFC. (A and B) Triple immunofluorescence staining in the mPFC showing PNN, Iba1, and iNOS ( A ) or PNN, Iba1, and Arg1 ( B ). OA rats showed increased PNN expression and pro-inflammatory (Iba1 + iNOS⁺) microglia, while HIIT reduced PNN levels and promoted anti-inflammatory (Iba1 + Arg1⁺) phenotypes. (C and D) Protein expression of iNOS, Arg1, IL-10, IL-1β, and TNF-α in the mPFC, detected by western blot ( C ) and quantified by bar graphs ( D ) ( n = 6). Statistical methods used: One-way ANOVA. ** p < 0.01 vs. the sham group, ## p < 0.01 vs. the OA group. Data are represented as mean ± SD.

Article Snippet: Equal amounts of protein (30–50 μg) were separated on 10–12% SDS-PAGE gels (FuturePAGE, ACE Biotechnology, ET15420LGel), transferred to PVDF membranes (Millipore, Billerica, MA, USA, IPVH00010), blocked with 5% skim milk (BD Biosciences, Franklin Lakes, NJ, USA) for 1 h, and incubated overnight at 4 °C with primary antibodies against β-actin (1:5000, Servicebio, GB15003-100), COL2A1 (1:1000, Santa Cruz Biotechnology, sc-52658), MMP13 (1:1000, Proteintech, 18165-1-AP), iNOS (1:1000, Santa Cruz Biotechnology, sc-7271), Arg1 (1:1000, Santa Cruz Biotechnology, sc-47715), IL-10 (1:1000, Santa Cruz Biotechnology, sc-32815), IL-1β (1:1000, Santa Cruz Biotechnology, sc-12742), and TNF-α (1:1000, Santa Cruz Biotechnology, sc-52746).

Techniques: Immunofluorescence, Staining, Expressing, Western Blot

Journal: Scientific Reports

Article Title: High-intensity interval training remodels perineuronal nets in the medial prefrontal cortex to drive microglial polarization and alleviate osteoarthritis pain

doi: 10.1038/s41598-026-40823-w

Figure Lengend Snippet: PNNs degradation and HIIT alleviate neuroinflammation in the mPFC of OA rats. ( A - D ) Representative triple immunofluorescence staining images of the mPFC, showing the distribution patterns of PNNs, Iba1 (microglial marker), and iNOS ( A ) or Arg1 ( C ). Quantification of panel A is shown in ( B ), and quantification of panel C is shown in ( D ) ( n = 6). ( E and F ) Western blot analysis and corresponding quantification of inflammatory markers, including iNOS, Arg1, IL-1β, TNF-α, and IL-10 in mPFC tissue lysates ( n = 6). Statistical methods used: One-way ANOVA. ** p < 0.01 vs. the OA+Vehicle group, ns not significant vs. the OA+ChABC group.Data are presented as mean ± SD.

Article Snippet: Equal amounts of protein (30–50 μg) were separated on 10–12% SDS-PAGE gels (FuturePAGE, ACE Biotechnology, ET15420LGel), transferred to PVDF membranes (Millipore, Billerica, MA, USA, IPVH00010), blocked with 5% skim milk (BD Biosciences, Franklin Lakes, NJ, USA) for 1 h, and incubated overnight at 4 °C with primary antibodies against β-actin (1:5000, Servicebio, GB15003-100), COL2A1 (1:1000, Santa Cruz Biotechnology, sc-52658), MMP13 (1:1000, Proteintech, 18165-1-AP), iNOS (1:1000, Santa Cruz Biotechnology, sc-7271), Arg1 (1:1000, Santa Cruz Biotechnology, sc-47715), IL-10 (1:1000, Santa Cruz Biotechnology, sc-32815), IL-1β (1:1000, Santa Cruz Biotechnology, sc-12742), and TNF-α (1:1000, Santa Cruz Biotechnology, sc-52746).

Techniques: Immunofluorescence, Staining, Marker, Western Blot

Journal: Scientific Reports

Article Title: High-intensity interval training remodels perineuronal nets in the medial prefrontal cortex to drive microglial polarization and alleviate osteoarthritis pain

doi: 10.1038/s41598-026-40823-w

Figure Lengend Snippet: PNN remodeling precedes microglial polarization. ( A - D ) Representative triple immunofluorescence staining images of the mPFC, showing PNN, Iba1, and iNOS ( A ), or PNN, Iba1, and Arg1 ( C ). Quantification of panel A is shown in ( B ), and quantification of panel C is shown in ( D ) ( n = 6). Statistical methods used: One-way ANOVA. ** p < 0.01. Data are presented as mean ± SD.

Article Snippet: Equal amounts of protein (30–50 μg) were separated on 10–12% SDS-PAGE gels (FuturePAGE, ACE Biotechnology, ET15420LGel), transferred to PVDF membranes (Millipore, Billerica, MA, USA, IPVH00010), blocked with 5% skim milk (BD Biosciences, Franklin Lakes, NJ, USA) for 1 h, and incubated overnight at 4 °C with primary antibodies against β-actin (1:5000, Servicebio, GB15003-100), COL2A1 (1:1000, Santa Cruz Biotechnology, sc-52658), MMP13 (1:1000, Proteintech, 18165-1-AP), iNOS (1:1000, Santa Cruz Biotechnology, sc-7271), Arg1 (1:1000, Santa Cruz Biotechnology, sc-47715), IL-10 (1:1000, Santa Cruz Biotechnology, sc-32815), IL-1β (1:1000, Santa Cruz Biotechnology, sc-12742), and TNF-α (1:1000, Santa Cruz Biotechnology, sc-52746).

Techniques: Immunofluorescence, Staining

Journal: European Journal of Medical Research

Article Title: Extracellular vesicles derived from endothelial cells modulate macrophage phenotype in vitro

doi: 10.1186/s40001-023-01427-6

Figure Lengend Snippet: iNOS expression in and CD86 expression on NR8383 macrophages upon 24 h stimulation with E-EVs. A Immunofluorescence analysis of the iNOS expression in NR8383 macrophages after stimulation with either plain PBS, E LPS -EVs, or E Nor -EVs. iNOS (green fluorescence) was detected in E LPS -EVs treated cells, while it could not be concluded in PBS nor E Nor -EVs treated cells. DAPI (blue fluorescence) indicates cellular nuclei. Used magnification was × 400. n = 3 replicates were used for these experiments. *p < 0.0001 for E LPS -EVs compared with the PBS and E Nor -EVs groups. Statistical analysis was performed by one‐way ANOVA with Bonferroni's correction. Scale bar: 30 µm. B Flow cytometry analysis of CD86 expression on NR8383 macrophages upon stimulation with either plain PBS, E LPS -EVs, or E Nor -EVs. CD86 expression significantly increased on NR8383 macrophages as a result of the E LPS -EVs stimulation when compared to the expression levels of the PBS and E Nor -EVs groups. CD86 expression quantification is represented as average in % + SEM (n = 3 replicates). *p < 0.0001 for E LPS -EVs compared with the PBS and E Nor -EVs groups. Statistical analysis was performed by one‐way ANOVA with Bonferroni's correction

Article Snippet: Antibodies iNOS-FITC (1:50, Novus Biologicals, USA) were used to identify NR8383 macrophages.

Techniques: Expressing, Immunofluorescence, Fluorescence, Flow Cytometry

Journal: International Journal of Immunopathology and Pharmacology

Article Title: Agmatine suppresses the imidazoline I 2 receptor/ribosomal S6 kinase 2/NF-κB signaling pathway regulating alveolar macrophage polarization and ameliorating sepsis-associated acute lung injury

doi: 10.1177/03946320261425360

Figure Lengend Snippet: The phenotype of alveolar macrophages in bronchoalveolar lavage fluid was detected by flow cytometry. CD11b + F4/80 + cells indicate macrophages. iNOS + cells indicate M1 macrophages. CD206 + cells indicate M2 macrophages. * p < 0.05. ** p < 0.01. *** p < 0.001.

Article Snippet: CD11b, F4/80, CD206, iNOS antibody, β-actin, ribosomal S6 kinase 2 (RSK2), phospho-RSK2 (p-RSK2), inhibitor of nuclear factor-κBα (IκBα), phospho-nuclear factor-κBα (p-IκBα), and phospho-P65 (p-p65) and secondary antibodies were purchased from CST, USA.

Techniques: Flow Cytometry

Journal: International Journal of Immunopathology and Pharmacology

Article Title: Agmatine suppresses the imidazoline I 2 receptor/ribosomal S6 kinase 2/NF-κB signaling pathway regulating alveolar macrophage polarization and ameliorating sepsis-associated acute lung injury

doi: 10.1177/03946320261425360

Figure Lengend Snippet: After Silencing RSK2 the lung tissue injury and the phenotype of alveolar macrophages in bronchoalveolar lavage fluid. (a, c) Histopathological changes were evaluated via H&E staining in each group. (b, d, e) The phenotype of alveolar macrophages in bronchoalveolar lavage fluid was detected by flow cytometry. CD11b + F4/80 + cells indicate macrophages. iNOS + cells indicate M1 macrophages. CD206 + cells indicate M2 macrophages. * p < 0.05. ** p < 0.01. *** p < 0.001.

Article Snippet: CD11b, F4/80, CD206, iNOS antibody, β-actin, ribosomal S6 kinase 2 (RSK2), phospho-RSK2 (p-RSK2), inhibitor of nuclear factor-κBα (IκBα), phospho-nuclear factor-κBα (p-IκBα), and phospho-P65 (p-p65) and secondary antibodies were purchased from CST, USA.

Techniques: Staining, Flow Cytometry

Journal: Antioxidants

Article Title: Placental Adaptive Changes to Protect Function and Decrease Oxidative Damage in Metabolically Healthy Maternal Obesity

doi: 10.3390/antiox9090794

Figure Lengend Snippet: NO-induced nitrosative stress markers in placental samples from normal weight (lean, n = 9) and obese (OB, n = 8) pregnancies. ( a ) iNOS expression levels. This enzyme was assessed in placental protein extracts by Western blot analysis, as described in the “Material and Methods” Section. ( b ) Detection and quantification of ERK nitrosylated (SNO-ERK1,2) levels. The placental extracts were processed and subjected to the Biotin-Switch method. The biotin-labeled SNO proteins were further subjected to neutravidin capture, separated by using 10% by SDS-PAGE, and immunoblotted with an anti-ERK antibody. Total ERK were represented as total protein (input). ( c ) Detection of nitrated proteins by immunoblot analysis using antinitrotyrosine antibodies. In each lane, all nitrated proteins were counted in order to quantify the total nitration protein level for each placental sample. Molecular weight markers (M) and sizes (kDa) are shown, and also possible targets of nitrated proteins (arrows) are indicated. In all cases: top represents the mean densitometry expressed in arbitrary units and bottom shows a representative picture of all Western blots. The results were expressed as means ± SD. * p < 0.05 relative to the lean group or input. iNOS: inducible nitric oxide synthase.

Article Snippet: Primary (anti-ERK1/2, anti-SOD 1, anti-catalase, and β-actin) and secondary antibodies came from Cell Signaling Technology (Danvers, MA, USA). iNOS antibody was from R&D Systems (Minneapolis, MN, USA) and nitrotyrosine antibody was from Merck Millipore (Darmstadt, Germany).

Techniques: Expressing, Western Blot, Labeling, SDS Page, Nitration, Molecular Weight