Journal: Biochimica et biophysica acta

Article Title: Redox regulation of platelet-derived-growth-factor-receptor: role of NADPH-oxidase and c-Src tyrosine kinase.

doi: 10.1016/j.bbamcr.2005.03.004

Figure Lengend Snippet: Fig. 3. Translocation of p47-phox and p-67phox to plasma membrane fraction after stimulation with PDGF or PMA. Starved NIH3T3 fibroblasts were stimulated or not with 20 ng/ml PDGF for 5 min or 250 nM PMA for 30 min. PDGF-stimulated cells were also pre-treated with 10 AM GF for 30 min. Western blotting of lysate membrane fraction with anti-p67-phox antibodies (A); after stripping procedure, the same filter was re-probed with anti-p47-phox antibodies (B), or with anti-PDGFr antibodies for the normalization (C). Bottom A and B: the normalized values, obtained by densitometric analysis, are reported as mean percentage of trans- locationFS.E. relative to the value obtained in non-stimulated cells (control, 100%) of three independent experiments. *P V0.05 compared to the control.

Article Snippet: Proteins were visualized by incubating the membranes with specific antibodies: mouse monoclonal anti-phosphotyrosine PY20 (anti-pTyr), rabbit polyclonal anti-PDGFr-h (anti-PDGFr), rabbit anti-c-Src, rabbit polyclonal anti-p47 phox or anti-p67 phox (Santa Cruz), rabbit anti-pTyr416 c-Src (BD).

Techniques: Translocation Assay, Clinical Proteomics, Membrane, Western Blot, Stripping Membranes, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Carbon Monoxide-Releasing Molecule-2 Ameliorates Particulate Matter-Induced Aorta Inflammation via Toll-Like Receptor/NADPH Oxidase/ROS/NF- κ B/IL-6 Inhibition

doi: 10.1155/2021/2855042

Figure Lengend Snippet: PM induces IL-6 expression via NADPH oxidase-derived ROS generation. (a) HASMCs were pretreated with either NAC (10 mM), DPI (1 μ M), or APO (100 μ M) for 2 h, followed by PM incubation for 24 h. The IL-6 secretion and mRNA levels were measured. Cells were treated with PM for designated times, and then the (b) ROS generation and (c) NADPH oxidase activity were determined. (d) Cells were incubated with PM for different intervals. The cytosolic and membrane fractions were prepared, and then the protein expression of p47 phox in each fraction was analyzed by Western blot (note: CE = cytosolic extraction; ME = membrane extraction). (e) Cells were transfected with siRNA of scrambled (scrb), p47 phox , or p67 phox , followed by PM incubation for 24 h. The IL-6 secretion and mRNA levels were measured. (f) Cells were transfected with siRNA of scrambled (scrb), p47 phox , TLR2, or TLR4, and subsequently incubated with PM for 2 h. The ROS generation and NADPH oxidase activity were determined. Data are presented as mean ± S.E.M. of three independent experiments. # P < 0.01, as compared with the cells exposed to PM alone (a) or PM plus scrb siRNA (e, f). # P < 0.01, as compared with control (b).

Article Snippet: In this study, human scrambled, TLR2, TLR4, p47 phox , p67 phox , p65, p50, and IL-6 siRNAs from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) were utilized.

Techniques: Expressing, Derivative Assay, Incubation, Activity Assay, Membrane, Western Blot, Extraction, Transfection, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Carbon Monoxide-Releasing Molecule-2 Ameliorates Particulate Matter-Induced Aorta Inflammation via Toll-Like Receptor/NADPH Oxidase/ROS/NF- κ B/IL-6 Inhibition

doi: 10.1155/2021/2855042

Figure Lengend Snippet: CORM-2 inhibits PM-induced ICAM-1 and VCAM-1 expression and monocyte adhesion in HASMCs. HASMCs were treated with PM for designated times, and then the VCAM-1 and ICAM-1 (a) protein expression and (b) mRNA levels were determined. (c) Cells were pretreated with NAC (10 mM), DPI (1 μ M), HLN (1 μ M), or CORM-2 (50 μ M) for 2 h, followed by PM incubation for 24 h. The VCAM-1 and ICAM-1 mRNA levels were measured. (d) Cells were transfected with siRNA of scrambled (scrb), TLR2, TLR4, p47 phox , p65, p50, or IL-6, and subsequently incubated with PM for 24 h. The VCAM-1 and ICAM-1 mRNA levels were analyzed. (e) Cells were pretreated with HLN (1 μ M), DPI (1 μ M), NAC (10 mM), CORM-2 (50 μ M), ICAM-1 neutralizing antibody (10 μ g/ml), or VCAM-1 neutralizing antibody (10 μ g/ml) for 2 h prior to incubation with PM for 24 h. The adherence of THP-1 monocytic cells was measured. All figures are representative of three independent experiments performed in duplicate. Data are presented as mean ± S.E.M. of three independent experiments. # P < 0.01, as compared with control (b). # P < 0.01, as compared with the cells exposed to PM alone (c) or PM plus scrb siRNA (d).

Article Snippet: In this study, human scrambled, TLR2, TLR4, p47 phox , p67 phox , p65, p50, and IL-6 siRNAs from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) were utilized.

Techniques: Expressing, Incubation, Transfection, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Carbon Monoxide-Releasing Molecule-2 Ameliorates Particulate Matter-Induced Aorta Inflammation via Toll-Like Receptor/NADPH Oxidase/ROS/NF- κ B/IL-6 Inhibition

doi: 10.1155/2021/2855042

Figure Lengend Snippet: PM induces MMP-2 and MMP-9 expression and cell migration via TLR2, TLR4, NADPH oxidase, NF- κ B, and IL-6 in HASMCs. (a, b) HASMCs were treated with PM for designated times, and then the MMP-2 and MMP-9 protein expression and mRNA levels and cell migration were determined. (c) Cells were transfected with siRNA of scrambled (scrb), TLR2, TLR4, p47 phox , p65, p50, or IL-6, followed by incubation with PM for 24 h. The MMP-2 and MMP-9 mRNA levels and cell migration were assessed. Data are presented as mean ± S.E.M. of three independent experiments. # P < 0.01, as compared with control (b). # P < 0.01, as compared with the cells exposed to PM plus scrb siRNA (c).

Article Snippet: In this study, human scrambled, TLR2, TLR4, p47 phox , p67 phox , p65, p50, and IL-6 siRNAs from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) were utilized.

Techniques: Expressing, Migration, Transfection, Incubation, Control

Journal: Frontiers in physiology

Article Title: Regulation of NADPH Oxidase-Mediated Superoxide Production by Acetylation and Deacetylation.

doi: 10.3389/fphys.2021.693702

Figure Lengend Snippet: FIGURE 6 | Rac1 deacetylation reduces its interaction with p67phox. (A): Recombinant Rac1 protein was incubated with p67phox, with or without SIRT1, in cell-free systems. Then, Rac1 pull-down was performed with glutathione- agarose beads. Western blot analyses were performed with antibodies against Rac1 or p67phox using the Rac1-pulldown samples. Panel (A) shows representative blots. (B) Acetylation of lysine 166 (K166) on Rac1 leads to conformational changes as revealed by in silico modeling. (C) Conformational changes induced by acetylation of K166 on Rac1 enhance its binding affinity for GTP. (D) K166 acetylation enhances the binding activity of Rac1 to p67phox as calculated by computational modeling. KD, dissociation constant.

Article Snippet: In vitro Rac1-p67phox Interaction and Rac1 Pull-Down Experiment The Rac1 deacetylation reaction mixtures (Rac1 ± SIRT1) were incubated with 3 μg recombinant p67phox (OriGene) at 4°C for 1 h. Then, Rac1 pull-down was performed with 40 μl 50% glutathione-agarose beads (BD Biosciences), as Rac1 was the only protein carrying a GST-tag in the reaction mixture.

Techniques: Recombinant, Incubation, Western Blot, In Silico, Binding Assay, Activity Assay

Journal: Redox Report : Communications in Free Radical Research

Article Title: Neutrophil extracellular traps drive osteoporosis via NCF2-dependent signaling: integrated transcriptomics with mechanistic validation

doi: 10.1080/13510002.2025.2534745

Figure Lengend Snippet: Integrative transcriptomic and experimental validation of NET formation in osteoporosis. Schematic overview of the study design. A rat model of postmenopausal osteoporosis (OVX) was established and subjected to transcriptomic analysis (RNA-seq) to identify differentially expressed genes (DEGs) enriched in neutrophil extracellular traps (NETs) formation pathways. Mechanistic insights into NETosis were explored, including activation of neutrophils, NOX complex assembly, and release of NETs containing NE, MPO, and CitH3. For in vitro validation, primary osteoblasts isolated from 7-day-old rats were treated with NET-conditioned medium derived from PMA-induced bone marrow neutrophils or HL-60–derived neutrophil-like cells. siRNA-mediated knockdown of NCF2 was performed to assess its role in NETs formation and subsequent osteogenic inhibition.

Article Snippet: The primary antibodies used included NE (Thermo Fisher, PA5-88916), MPO (Thermo Fisher, MPO-101AP), VDAC1 (Novus Biologicals, NBP3-15871), PLCG2 (Novus Biologicals, NBP1-86030), NCF2 (Novus Biologicals, NBP3-16257), and β-actin (Proteintech, 66009-1-Ig).

Techniques: Biomarker Discovery, RNA Sequencing, Activation Assay, In Vitro, Isolation, Derivative Assay, Knockdown, Inhibition

Journal: Redox Report : Communications in Free Radical Research

Article Title: Neutrophil extracellular traps drive osteoporosis via NCF2-dependent signaling: integrated transcriptomics with mechanistic validation

doi: 10.1080/13510002.2025.2534745

Figure Lengend Snippet: SMR discovery and experimental validation of NETs-related biomarkers in osteoporosis. A, Forest plot summarizing SMR results for VDAC1, PLCG2 and NCF2 (β, SE, P, HEIDI P, and OR ± 95% CI). B, Representative IHC images of distal femur (brown = positive DAB; hematoxylin counter-stain) showing increased staining for the three proteins in OVX rats versus Sham; scale bar = 50 µm. C, Western blots of neutrophil lysates (Control vs PMA-induced NETs) probed for NCF2 (65 kDa), VDAC1 (31 kDa) and PLCG2 (148 kDa); GAPDH (36 kDa) serves as loading control. D–F, Quantification of IHC-positive area (% of marrow) for each protein (mean ± SD). G–I, Densitometric ratios of target proteins to GAPDH in western blots (mean ± SD). P < 0.05*, < 0.01 **, < 0.001 ***, < 0.0001 **** (two-tailed t-test).

Article Snippet: The primary antibodies used included NE (Thermo Fisher, PA5-88916), MPO (Thermo Fisher, MPO-101AP), VDAC1 (Novus Biologicals, NBP3-15871), PLCG2 (Novus Biologicals, NBP1-86030), NCF2 (Novus Biologicals, NBP3-16257), and β-actin (Proteintech, 66009-1-Ig).

Techniques: Biomarker Discovery, Staining, Western Blot, Control, Two Tailed Test

Journal: Redox Report : Communications in Free Radical Research

Article Title: Neutrophil extracellular traps drive osteoporosis via NCF2-dependent signaling: integrated transcriptomics with mechanistic validation

doi: 10.1080/13510002.2025.2534745

Figure Lengend Snippet: Knockdown of NCF2 decreases NETS formation and reverses NETs-mediated suppression of osteoblast differentiation. (A) Representative Western blot confirming NCF2 knockdown efficiency in HL-60 neutrophil-like cells. (B, C) Quantification of NCF2 protein and mRNA expression levels (**** P < 0.0001). (D) Triple immunofluorescence staining (DAPI, NE, MPO, CitH3) illustrating reduced NETS formation upon NCF2 knockdown; scale bar = 50 µm. (E–G) Quantitative analysis of NE-, MPO-, and CitH3-positive areas (**** P < 0.0001). (H) Extracellular DNA concentrations in supernatants measured by UV spectrophotometry (**** P < 0.0001). (I, J) ALP staining at day 7 (scale bar = 200 µm) and Alizarin red S staining of mineralisation at day 21 (scale bar = 200 µm) after exposure to indicated conditioned media. (K–M) Western blot and quantitative densitometric analysis of ALP and OCN protein expression levels normalised to GAPDH (*** P < 0.001, **** P < 0.0001). Data represent mean ± SD of three independent experiments; statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: The primary antibodies used included NE (Thermo Fisher, PA5-88916), MPO (Thermo Fisher, MPO-101AP), VDAC1 (Novus Biologicals, NBP3-15871), PLCG2 (Novus Biologicals, NBP1-86030), NCF2 (Novus Biologicals, NBP3-16257), and β-actin (Proteintech, 66009-1-Ig).

Techniques: Knockdown, Western Blot, Expressing, Immunofluorescence, Staining, Spectrophotometry

Journal: Redox Report : Communications in Free Radical Research

Article Title: Neutrophil extracellular traps drive osteoporosis via NCF2-dependent signaling: integrated transcriptomics with mechanistic validation

doi: 10.1080/13510002.2025.2534745

Figure Lengend Snippet: Docking results of available proteins small molecules. (A) NCF2 docking Diltiazem. (B) VDAC1 docking Diltiazem. (C) PLCG2 docking Exemestane. (D) NCF2 docking Cimetidine. (E) VDAC1 docking Cimetidine. (F) NCF2 docking Desipramine. (G) NCF2 docking Profenamine. (H) VDAC1 docking Emodin. (I) VDAC1 docking Tolazoline. (J) VDAC1 docking Zuclopenthixol. (K) VDAC1 docking Ethynyl estradiol. (L) PLCG2 docking Simvastatin.

Article Snippet: The primary antibodies used included NE (Thermo Fisher, PA5-88916), MPO (Thermo Fisher, MPO-101AP), VDAC1 (Novus Biologicals, NBP3-15871), PLCG2 (Novus Biologicals, NBP1-86030), NCF2 (Novus Biologicals, NBP3-16257), and β-actin (Proteintech, 66009-1-Ig).

Techniques:

Journal: Scientific reports

Article Title: Candida albicans aspartyl protease (Sap6) inhibits neutrophil function via a "Trojan horse" mechanism.

doi: 10.1038/s41598-025-91425-x

Figure Lengend Snippet: Fig. 7. Degradation of NADPH oxidase subunits. To determine the ability of Sap6 to degrade selected subunits of NADPH oxidase, neutrophils (1 × 106 cells/sample) were incubated with Sap6 at a concentration of 1200 ng/ml for 1 h and then lysed. The lysates were subjected to electrophoretic separation and transferred to a membrane, after which the (a) p67phox and (b) gp91phox subunits were identified using antibodies. The graphs show the intensities of the bands corresponding to the degradation products of the individual oxidase subunits. (c) To evaluate the capacity of Sap6 to degrade the p67phox subunit, an experiment was conducted with the isolated protein. The oxidase subunit was incubated with Sap6 at a substrate-to-enzyme ratio of 50:1 for 3 h. This was followed by electrophoretic separation and subsequent staining to visualize the degradation products. As a control to gauge the proteolytic activity of the enzyme, a sample was incubated with a protease inhibitor (pepA) for 30 min prior to the addition of Sap6.

Article Snippet: The membranes were blocked in TBST (25 mM Tris, pH 7.2; 150 mM NaCl; 0.1% Tween) with 3% nonfat dry milk for 1 h at room temperature and incubated overnight at 4 °C in blocking buffer with an anti-p67phox antibody (R&D Systems, Minneapolis, MN, USA) (1:400) or an anti-gp91phox antibody (Abcam) (1:1000).

Techniques: Incubation, Concentration Assay, Membrane, Isolation, Staining, Control, Activity Assay, Protease Inhibitor

Journal: The Journal of Experimental Medicine

Article Title: The phosphoinositide-binding protein p40 phox activates the NADPH oxidase during FcγIIA receptor–induced phagocytosis

doi: 10.1084/jem.20052085

Figure Lengend Snippet: Expression of FcγIIA receptor, phagocytosis, and NADPH oxidase activity. (A) Analysis of FcγIIA expression by flow cytometry using an FITC-conjugated antibody against CD32 is shown for COS7 and COS phox FcγR cells (left) and human peripheral blood monocytes (right) as indicated. The gray histogram indicates staining of either COS phox FcγR or monocytes with an FITC-conjugated isotype control mAb. (B and C) Phagocytosis of IgG–sheep RBCs in media containing NBT. (B) COS phox FcγR cells incubated with IgG-RBCs for 30 min at 37°C. Many of the ingested IgG-RBCs, which appear tan, are indicated by arrows. (C) Murine bone marrow–derived macrophages incubated with IgG-RBCs for 10 min at 37°C. Formazan-stained phagosomes, indicative of intraphagosomal superoxide production, are indicated by arrows. (D) COS phox FcγR cells incubated with 100 ng/ml phorbol myristate acetate for 30 min, showing diffuse formazan deposits. Bar, 30 μm.

Article Snippet: A rabbit polyclonal antibody against p40 phox and a mouse mAb against Rac were from Upstate Biotechnology, and mAbs against p67 phox and p47 phox were from BD Biosciences.

Techniques: Expressing, Activity Assay, Flow Cytometry, Staining, Incubation, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: The phosphoinositide-binding protein p40 phox activates the NADPH oxidase during FcγIIA receptor–induced phagocytosis

doi: 10.1084/jem.20052085

Figure Lengend Snippet: Expression of p40 phox in COS phox FcγR cells and FcγR-elicited NADPH oxidase activity. Data shown is representative of at least three independent experiments. (A) Immunoblots of human neutrophil and COS7 cell lysates (10 μg protein per lane) probed with antibodies for p40 phox , p47 phox , and p67 phox . COS phox FcγR cells were transfected with either a stable p40 phox transgene or with varying amounts of p40pRK5 for transient expression as indicated. (B) IgG-RBC–elicited NADPH oxidase activity in COS phox FcγR cells expressing p40 phox . Multiple formazan-stained phagosomes (arrows) in COS phox FcγR transfected with 0.05 μg p40pRK5 (representative photomicrograph; bar, 30 μm). Similar numbers of formazan-stained phagosomes were present in COS phox FcγR cells transfected with larger amounts of plasmid or expressing p40 phox from a stable transgene. (C) Flow cytometry analysis of COSFcγR cell lines incubated with Fc OxyBURST Green–opsonized zymosan for 30 min. Fluorescence intensity is shown on the x axis.

Article Snippet: A rabbit polyclonal antibody against p40 phox and a mouse mAb against Rac were from Upstate Biotechnology, and mAbs against p67 phox and p47 phox were from BD Biosciences.

Techniques: Expressing, Activity Assay, Western Blot, Transfection, Staining, Plasmid Preparation, Flow Cytometry, Incubation, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: The phosphoinositide-binding protein p40 phox activates the NADPH oxidase during FcγIIA receptor–induced phagocytosis

doi: 10.1084/jem.20052085

Figure Lengend Snippet: Expression of p40 phox mutants in COS phox FcγR cells and effect on IgG–sheep RBC–elicited NADPH oxidase activity. Data shown is representative of at least three independent experiments. (A) Immunoblot of cell lysates from COS phox FcγR cells transfected with 0.67 μg of either empty pRK5 or pRK5 containing cDNAs for either wild-type or mutant p40 phox . Blots were probed with antibodies for p40 phox , p47 phox , and p67 phox . (B) COS phox FcγR cells were transfected as in A and incubated with IgG-RBCs in the presence of NBT for 30 min at 37°C. The percentage of cells with NBT + phagosomes is shown as the mean ± SD ( n = 4 except for W207R/D289A, where n = 3). (C) COS phox FcγR cells were transfected as in A for expression of YFP-tagged wild-type or mutant derivatives of p40 phox as indicated or a YFP-tagged PX domain of p40 phox and incubated with IgG–sheep RBCs or with IgG latex beads (*) without or with 50 nM wortmannin, followed by confocal microscopy. Individual phagosomes were scored for either the presence (black bars) or absence of YFP-p40 phox or YFP-p40PX translocation. The number of phagosomes scored for each construct is also shown. Data was collected from two to four independent experiments.

Article Snippet: A rabbit polyclonal antibody against p40 phox and a mouse mAb against Rac were from Upstate Biotechnology, and mAbs against p67 phox and p47 phox were from BD Biosciences.

Techniques: Expressing, Activity Assay, Western Blot, Transfection, Mutagenesis, Incubation, Confocal Microscopy, Translocation Assay, Construct

Journal: The Journal of Experimental Medicine

Article Title: The phosphoinositide-binding protein p40 phox activates the NADPH oxidase during FcγIIA receptor–induced phagocytosis

doi: 10.1084/jem.20052085

Figure Lengend Snippet: Interactions between p47 phox , p67 phox , and p40 phox subunits of the phagocyte NADPH oxidase. Structural motifs and identified interactions between p47 phox , p67 phox , and p40 phox are shown schematically. The p47 phox subunit contains a PX domain, two SH3 domains, and a C-terminal PRR. A domain containing four tetratricopeptide repeat (TPR) motifs comprises the N terminus of p67 phox , followed by an SH3, PB1, and second SH3 domain. The p67 phox subunit also contains a PRR adjacent to the N-terminal SH3 domain. p40 phox also contains a PX and PB1 domain, along with an intervening SH3 domain. In the p47 phox –p67 phox –p40 phox complex, p47 phox associates with p67 phox via a high-affinity tail-to-tail interaction involving the C-terminal PRR and SH3 domains in p47 phox and p67 phox , respectively, whereas p40 phox is tethered to p67 phox via a back-to-front interaction between their PB1 domains.

Article Snippet: A rabbit polyclonal antibody against p40 phox and a mouse mAb against Rac were from Upstate Biotechnology, and mAbs against p67 phox and p47 phox were from BD Biosciences.

Techniques: