Journal: Redox Biology

Article Title: Redox regulation of lung endothelial PERK, unfolded protein response (UPR) and proliferation via NOX1: Targeted inhibition as a potential therapy for PAH

doi: 10.1016/j.redox.2025.103554

Figure Lengend Snippet: Non-Standard Abbreviations and Acronyms

Article Snippet: The primary antibodies and subsequent dilutions used in this study include: mouse anti-β-actin (Santa Cruz, sc-47778 1:2500), rabbit anti-HIF1α (Abcam, ab179483, 1:500), rabbit anti-NOX1 (Abcam, ab131088, 1:500), rabbit anti-NOXO1 (ThermoFisher, 600-401-899, 1:1000), rabbit anti-NOXA1 (Abclonal, A13844, 1:500), rabbit anti-NOX2 (Abcam, ab80508, 1:500), rabbit anti-NOX4 (Abcam, ab133303, 1:500), goat anti-p22 phox (Santa Cruz, sc-11712, 1:1000), rabbit anti-p47 phox (Millipore Sigma, 07-001, 1:500), goat anti-p67 phox (Santa Cruz, sc-7663, 1:1000), rabbit anti-phospho-eIF2α (Cell Signaling, 9721, 1:500), rabbit anti-eIF2α (Cell Signaling, 5324, 1:500), rabbit anti-BiP (Cell signaling, 3177, 1:1000), rabbit anti-PDI (Cell signaling, 3501, 1:1000), rabbit anti-ATF4 (Cell signaling, 11815, 1:1000), rabbit anti-phospho-PERK (Cell signaling, 3179, 1:1000), rabbit anti-PERK (Cell signaling, 3192, 1:1000).

Techniques: Binding Assay, Formalin-fixed Paraffin-Embedded, Gene Expression, Membrane

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Loss of endogenous Nox2-NADPH oxidase does not prevent age-induced platelet activation and arterial thrombosis in mice

doi: 10.1016/j.rpth.2024.102597

Figure Lengend Snippet: mRNA for Nox2 is increased in platelets from aged mice. Levels of mRNA were measured in bead-purified platelets using quantitative polymerase chain reaction for catalytic subunits (A) Nox2, (B) Nox4, and (C) Nox1, and regulatory subunits (D) p47Phox and (E) p67Phox of NADPH oxidase and for Nox2-associated subunit (F) p22Phox. Relative expression to young Nox2 wild-type (Nox2-WT) is presented. Data are presented as median with IQR and analyzed using the Kruskal–Wallis test with Dunn’s multiple comparisons. N = 6 per group. Nox2-KO, Nox2 knockout.

Article Snippet: Samples containing 20 μg of protein were run on a 12% sodium dodecyl sulfate gel and transferred to a PVDF membrane; membranes were blocked using 5% filtered BSA and then stained overnight at 4 °C with primary antibodies for Nox1 (Novus Biosciences, NBP1-31546), Nox4 (Novus Biosciences, NB110–58851), p47phox (Santa Cruz, sc-17844), p67phox (Santa Cruz, sc-374510), or β-actin (Santacruz, Sc-47778) and blots were developed using standard procedure [ ].

Techniques: Purification, Real-time Polymerase Chain Reaction, Expressing, Knock-Out

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Loss of endogenous Nox2-NADPH oxidase does not prevent age-induced platelet activation and arterial thrombosis in mice

doi: 10.1016/j.rpth.2024.102597

Figure Lengend Snippet: Protein levels of NADPH oxidase subunits or enzymatic activity in platelets are not altered by Nox2 genotype or age. Protein levels were measured in bead-purified platelets by Western blotting for catalytic subunits (A) Nox1 and (B) Nox4 and regulatory subunits (C) p47Phox and (D) p67Phox of NADPH oxidase. The activity of NADPH oxidase was measured in platelet lysate in the presence of cytochrome C and NADPH, and changes in absorbance in the presence or absence of superoxide dismutase were calculated and expressed as the amount of superoxide generated. Data are presented as median with IQR for A and C and as mean ± SE for B, D, and E. Data were analyzed using the Kruskal–Wallis test with Dunn’s multiple comparisons. N = 4 to 8 per group. AKO; aged Nox2 knockout; AW; aged Nox2 wild-type; Nox2-KO, Nox2 knockout; Nox2-WT, Nox2 wild-type; YKO; young Nox2 knockout; YW, young Nox2 wild-type.

Article Snippet: Samples containing 20 μg of protein were run on a 12% sodium dodecyl sulfate gel and transferred to a PVDF membrane; membranes were blocked using 5% filtered BSA and then stained overnight at 4 °C with primary antibodies for Nox1 (Novus Biosciences, NBP1-31546), Nox4 (Novus Biosciences, NB110–58851), p47phox (Santa Cruz, sc-17844), p67phox (Santa Cruz, sc-374510), or β-actin (Santacruz, Sc-47778) and blots were developed using standard procedure [ ].

Techniques: Activity Assay, Purification, Western Blot, Generated, Knock-Out

Journal: Redox Biology

Article Title: Cathepsin C from extracellular histone-induced M1 alveolar macrophages promotes NETosis during lung ischemia-reperfusion injury

doi: 10.1016/j.redox.2024.103231

Figure Lengend Snippet: CTSC activates p38 MAPK and results in p47 phox phosphorylation and trafficking of intracellular NADPH oxidase subunits to the cell membrane. a. Protein levels of p-p38 and p38 in PMNs treated with CTSC were detected using western blot (n = 3 per group). b. The activity of NADPH oxidase was estimated using the WST-8 method, indicated by the NADP + /NADPH ratio (n = 3 per group). c. Representative images and quantitative analysis of staining for DCFH-DA in PMNs treated with CTSC and SB203580 (n = 3 per group). Scale Bar = 50 μm. d-f. Representative images and quantitative analysis of NET formation (Scale Bar = 20 μm), protein levels of PAD4 and CiH3 (n = 3 per group), and concentrations of dsDNA in the supernatant (n = 3 per group) of PMNs pretreated with CTSC and SB203580 were presented. g. Protein levels of p-p38 and p38 in PMNs after treated with CTSC and inhibitors of ROS or NADPH oxidase were detected (n = 3 per group). h. Schematic illustrations depicting the structure of NADPH oxidase in both resting and activated states. i. Protein levels of p67 phox and p47 phox in both the plasma membrane and the whole cell in PMNs treated with CTSC were detected using western blot. Relative intensities were determined and normalized to the band intensity of GAPDH and Na + /K + -ATPase (n = 3 per group). j. Protein levels of p-p47 phox and p47 phox in the whole cell, as well as p67 phox and p47 phox in the plasma membrane of PMNs treated with CTSC and SB203580 were detected using western blot. Relative intensities were determined and normalized to the band intensity of GAPDH and Na + /K + -ATPase (n = 3 per group). Graphs represent means ± standard deviations. Statistical significance is indicated as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, and ns signifies P ＞ 0.05 when comparing data between two groups.

Article Snippet: Proteins in PMNs were incubated with anti-p38 mitogen-activated protein kinase (MAPK) (diluted at 1:1000; #8690T; CST, USA), anti-phospho-p38 MAPK (diluted at 1:1000; #4511T; CST, USA), anti-p67 phox (diluted at 1:5000; ab109523; Abcam, UK), anti-p47 phox (diluted at 1:500; A5143; ABclonal, China) and anti-phospho-p47 phox (diluted at 1:500; ab63554; Abcam, UK).

Techniques: Membrane, Western Blot, Activity Assay, Staining