Journal: Journal of Molecular Signaling

Article Title: NADPH oxidase mediates the oxygen-glucose deprivation/reperfusion-induced increase in the tyrosine phosphorylation of the N -methyl-D-aspartate receptor NR2A subunit in retinoic acid differentiated SH-SY5Y Cells

doi: 10.1186/1750-2187-7-15

Figure Lengend Snippet: Increased ROS generation during reperfusion of OGD subjected cultures of retinoic acid differentiated SH-SY5Y involves NADPH oxidase activity. Representative fluorescent images from three independent experiments of reactive oxygen species production following OGD ( A ) with vehicle (1:1000 DMSO) or DPI (100 nM) detected using dihydroethidium (DHE). Spectrophotometric quantification of reactive oxygen species following OGD ( B ) with vehicle (1:1000 DMSO) or DPI (100 nM) utilizing nitrobluetetrazolium chloride (NBT). ( C ) Western blot shown is representative of three independent experiments comparing p67 phox , NR2A subunit protein expression in non- and differentiated SH-SY5Y cells. ( D ) Quantification of the relative densitometry of p67 phox immunoreactive band in non- and differentiated SH-SY5Y cells. Data represent fold change of OGD/R groups versus normoxic control group (arbitrary units) ± S.E.M from three separate experiments that consisted of at least 6 determinents (askteriks * indicates a p <0.05 from vehicle treated normoxic control; ANOVA with post hoc Bonferroni test). Phorbol 12-myristate 13-acetate (PMA; 1 μM for 15 minutes) was used as a positive control for NADPH oxidase activity in both the DHE and NBT assays. Quantitative data from p67 phox protein expression represent fold change of differentiated SH-SY5Y compared to non-differentiated SH-SY5Y cells (arbitrary units) ± S.E.M from three separate experiments (askteriks * indicates a p <0.01 from non-differentiated control; student t -Test).

Article Snippet: The affinity purified rabbit-polyclonal p67 phox antibody (1:1000) was purchased from Millipore (Billerica, MA, USA).

Techniques: Activity Assay, Western Blot, Expressing, Control, Positive Control

Journal: PLoS ONE

Article Title: Administration of L-arginine plus L-citrulline or L-citrulline alone successfully retarded endothelial senescence

doi: 10.1371/journal.pone.0192252

Figure Lengend Snippet: Cells were incubated with NG or HG for 3 days with or without L-Arg (300 μM) or L-Cit (300 μM). (A) ROS generation was detected as intracellular oxidant generation by flow cytometry (n = 6). (B) Typical western blots for p22 phox , p47 phox and p67 phox , a subunit of NADPH oxidase, are shown. (C-E) A summary of the quantification of densitometric measurements of the immunoblot data. The data were normalized to ß-actin (n = 3–5). ##P<0.01, #P<0.05 versus NG. **P<0.01, *P<0.05 versus HG. Data are given as the mean ± SD from at least three independent experiments.

Article Snippet: We used the following commercially available primary antibodies: anti-human eNOS (#610297, BD Bioscience, USA) mouse monoclonal antibody (1:2,000), anti-human phosphorylated-eNOS (Ser-1177) (sc-12972, Santa Cruz, USA) rabbit polyclonal antibody (1:300), anti-human p22 phox (sc-20781, Santa Cruz, USA) rabbit polyclonal antibody (1:300), anti-human p16 INK4a (ab54210, Abcam, UK) mouse monoclonal antibody (1:200), anti-human arginase 2 (sc-20151, Santa Cruz, USA) rabbit polyclonal antibody (1:1000), anti-human p47 phox (sc-17845, Santa Cruz, USA) mouse monoclonal antibody (1:200), anti-human p67 phox (sc-384510, Santa Cruz, USA) mouse monoclonal antibody (1:200), and anti-human ß-actin (Abcam, Cambridge, UK) mouse monoclonal antibody (1:15,000).

Techniques: Incubation, Flow Cytometry, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Human Tissue Kallikrein 1 Is Downregulated in Elderly Human Prostates and Possesses Potential In Vitro Antioxidative and Antifibrotic Effects in Rodent Prostates

doi: 10.1155/2021/8877540

Figure Lengend Snippet: Pathological changes and oxidative status of prostate in aged wild rats and aged human KLK1 transgenic rats. (a, b) Representative HE photos and Masson photos in rat prostates (magnification ×100). (c) The area ratio of collagen in the Masson photos of rat prostates. (d, f) Representative Western blot results of Collagen I, Collagen III, NOX2, NOX4, p47 phox , and p67 phox in prostates of all three groups. (e, g) The expression levels of above-mentioned proteins with β -actin as the loading control in all three groups. (h, i) MDA content and SOD activity normalized to total protein concentration of rat prostate samples. For each group, values are presented as the mean ± SD of 10 rats per group. ∗ P < 0.05, ∗∗ P < 0.01 (aTGR vs. aWTR). ## P < 0.01 (aWTR vs. yWTR). §§ P < 0.01 (aTGR vs. yWTR).

Article Snippet: In Western blot experiments, the primary antibodies against rat KLK1 (Catalog No. ab131029) and human KLK1 (Catalog No. ab28289) were purchased from Abcam (USA); the primary antibodies against NADPH oxidase 2 (NOX2) (Catalog No. 19013-1-AP), NOX4 (Catalog No. 14347-1-AP), p47 phox (Catalog No. 28187-1-AP), p67 phox (Catalog No. 15551-1-AP), Collagen I (Catalog No. 14695-1-AP), Collagen III (Catalog No. 22734-1-AP), α -SMA (Catalog No. 14395-1-AP), DDAH2 (Catalog No. 14966-1-AP), TGF- β 1 (Catalog No. 21898-1-AP), RhoA (Catalog No. 10749-1-AP), ROCK1 (Catalog No. 21850-1-AP), COX-2 (Catalog No. 12375-1-AP), and β -actin (Catalog No. 20536-1-AP) were purchased from Proteintech (China); the primary antibodies against PTGIS (Catalog No. DF4745), inducible NO synthase (iNOS) (Catalog No. AF0199), eNOS (Catalog No. AF0096), β -Tubulin (Catalog No. T0023), and the secondary antibodies were from Affinity (USA).

Techniques: Transgenic Assay, Western Blot, Expressing, Activity Assay, Protein Concentration