Journal: Redox Biology

Article Title: p47 phox deficiency impairs platelet function and protects mice against arterial and venous thrombosis

doi: 10.1016/j.redox.2020.101569

Figure Lengend Snippet: ROS generation and phosphorylation of VASP, ERK1/2, p38 MAPK, ERK5, JNK, AKT and c-PLA2. (A) Western blot analysis of the expression of NOX2, p67 phox , NOX1, NOXO1 and Rac in WT and p47 phox -/- platelets. (B) ROS generation in platelets after stimulation with CRP (2 μg/ml) or thrombin (0.5 U/ml) was expressed as mean fluorescent intensity (MFI) (mean ± SE, n = 6) (Student t-test). (C) The phosphorylation level of VASP, ERK1/2, p38, ERK5 and JNK in CRP-stimulated platelets was detected by western blot and (D) quantified as a ratio relative to the total level (mean ± SD, n = 3) (Two-way ANOVA). (E) The phosphorylation level of AKT and c-PLA2 was also detected and (F) quantified (mean ± SD, n = 3) (Two-way ANOVA). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Immunoblotting assay involves antibodies against p47 phox (Santa Cruz Biotechnology); NOX1 and NOX2 (Proteintech); p67 phox (Abcam); NOXO1 (ABclonal Technology); Rac (Santa Cruz Biotechnology); Syk (anti-Tyr-525 and pan-Syk, Bioworld Technology); PLCγ2 (anti-Tyr-1217 and pan-PLCγ2; Bioworld Technology); VASP (anti-Ser157, Affinity Biosciences; anti-Ser239 and pan-VASP, Cell Signaling Technology); ERK1/2 (anti-Thr202/Tyr204 and pan-ERK1/2, Cell Signaling Technology); p38 MAPK (anti-Thr180/Tyr182, Cell Signaling Technology); ERK5 (anti-Thr218/Tyr220, Cell Signaling Technology; pan-ERK5, Proteintech); JNK (anti-Thr183/Tyr185, Cell Signaling Technology; pan-JNK, Affinity Biosciences); AKT (anti-Thr308, Cell Signaling Technology; pan-AKT, Affinity Biosciences); c-PLA2 (anti-Ser505 and pan-c-PLA2, Affinity Biosciences); NOX1 (Novus Biologicals); NOX2 (Abcam).

Techniques: Western Blot, Expressing

Journal: Redox Biology

Article Title: p47 phox deficiency impairs platelet function and protects mice against arterial and venous thrombosis

doi: 10.1016/j.redox.2020.101569

Figure Lengend Snippet: p47 phox translocation and interaction with NOX1 and NOX2. (A) Platelets were treated with CRP (5 μg/ml) for 30s or thrombin (1 U/ml) for 60s followed by isolation of the membrane and cytosol to measure the expression of p47 phox and (B) p47 phox expression was quantified as a ratio relative to the internal control β3 or GAPDH. Data were presented as mean ± SD (n = 3). Compared to corresponding 0, *p < 0.05. (C) Immunoprecipitation analysis of the interaction of p47 phox with NOX2 or NOX1 in platelets after stimulation with CRP (5 μg/ml) or thrombin (1 U/ml). Compared to resting, *p < 0.05; **p < 0.01; ***p < 0.001. (D) ROS generation in p47 phox -/- platelets in the presence of ML171 (5 μM) (NOX1 inhibitor) and/or gp91ds-at (50 μM) (NOX2 inhibitor) after CRP (2 μg/ml) or thrombin (0.5 U/ml) stimulation. (E) Platelet aggregation in p47 phox -/- platelets in the presence of ML171 (5 μM) (NOX1 inhibitor) and/or gp91ds-at (50 μM) (NOX2 inhibitor) after CRP (0.25 μg/ml) stimulation. Data were presented as mean ± SE (n = 6–8) (One-way ANOVA). Compared to WT, *p < 0.05; **p < 0.01. Compared with p47 phox -/- , &p < 0.01.

Article Snippet: Immunoblotting assay involves antibodies against p47 phox (Santa Cruz Biotechnology); NOX1 and NOX2 (Proteintech); p67 phox (Abcam); NOXO1 (ABclonal Technology); Rac (Santa Cruz Biotechnology); Syk (anti-Tyr-525 and pan-Syk, Bioworld Technology); PLCγ2 (anti-Tyr-1217 and pan-PLCγ2; Bioworld Technology); VASP (anti-Ser157, Affinity Biosciences; anti-Ser239 and pan-VASP, Cell Signaling Technology); ERK1/2 (anti-Thr202/Tyr204 and pan-ERK1/2, Cell Signaling Technology); p38 MAPK (anti-Thr180/Tyr182, Cell Signaling Technology); ERK5 (anti-Thr218/Tyr220, Cell Signaling Technology; pan-ERK5, Proteintech); JNK (anti-Thr183/Tyr185, Cell Signaling Technology; pan-JNK, Affinity Biosciences); AKT (anti-Thr308, Cell Signaling Technology; pan-AKT, Affinity Biosciences); c-PLA2 (anti-Ser505 and pan-c-PLA2, Affinity Biosciences); NOX1 (Novus Biologicals); NOX2 (Abcam).

Techniques: Translocation Assay, Isolation, Expressing, Immunoprecipitation

Journal: Journal of Animal Science and Biotechnology

Article Title: Maternal supply of cysteamine alleviates oxidative stress and enhances angiogenesis in porcine placenta

doi: 10.1186/s40104-021-00609-8

Figure Lengend Snippet: Effects of oxidative stress induced by H 2 O 2 on the Stat3/VEGF-A pathway in porcine vascular endothelial cells (PVECs). A The mRNA expression of angiogenesis-related factors (vascular endothelial growth factor A, VEGF-A ; interleukin-6/8, IL-6/8 ; NADPH oxidase 2, NOX2 ). B Schematic for the mechanism of H 2 O 2 -induced angiogenesis impairment. C , D Western blotting analysis of the expression of phospho-Stat3 (p-Stat3), NOX2, and VEGF-A. Cells were treated with 200 μmol/L H 2 O 2 for 24 h. Data are presented as mean ± SEM ( n = 3). Different letters indicate significant differences at P < 0.05

Article Snippet: After blocking with TBS/T buffer containing 5% milk, the membranes were incubated with the primary antibodies against vascular endothelial growth factor A (VEGF-A) (19003–1-AP, Proteintech, USA, 1:1,000), NADPH oxidase 2 (NOX2) (19013–1-AP, Proteintech, USA, 1:1,000), signal transducer and activator of transcription-3 (Stat3)(ab76315, Abcam, USA, 1:1,500), p-Stat3 (ab68153, Abcam, USA, 1:1,500), and β-actin (4970, CST, USA, 1:1,000).

Techniques: Expressing, Western Blot

Journal: Journal of Animal Science and Biotechnology

Article Title: Maternal supply of cysteamine alleviates oxidative stress and enhances angiogenesis in porcine placenta

doi: 10.1186/s40104-021-00609-8

Figure Lengend Snippet: Cysteamine (CS) pretreatment prolongs the phosphorylation of Stat3 in H 2 O 2 -treated PVECs. A , B Western blotting analysis of the expression of Phospho-Stat3 (p-Stat3), NADPH oxidase 2 (NOX2), and vascular endothelial growth factor A (VEGF-A). Cells were pretreated with various concentrations of cysteamine (0.5, 1 or 2 mmol/L CS) for 2 h, and then challenged with H 2 O 2 (200 μmol/L) for 24 h ( n = 3). C , D Representative images of tube formation by PVECs, pretreated with CS (0.5 mmol/L) and/or inhibitors of Stat3 (5 μmol/L stattic) for 2 h, and then challenged with H 2 O 2 (200 μmol/L) for 24 h ( n = 5; bar = 100 μm)5. E CCK8 assay was used to measure cell viability after different treatments as described above ( n = 6). F , G Western blotting analysis of the expression of p-Stat3 and VEGF-A ( n = 3). Data are presented as mean ± SEM. Different letters indicate significant differences at P < 0.05

Article Snippet: After blocking with TBS/T buffer containing 5% milk, the membranes were incubated with the primary antibodies against vascular endothelial growth factor A (VEGF-A) (19003–1-AP, Proteintech, USA, 1:1,000), NADPH oxidase 2 (NOX2) (19013–1-AP, Proteintech, USA, 1:1,000), signal transducer and activator of transcription-3 (Stat3)(ab76315, Abcam, USA, 1:1,500), p-Stat3 (ab68153, Abcam, USA, 1:1,500), and β-actin (4970, CST, USA, 1:1,000).

Techniques: Western Blot, Expressing, CCK-8 Assay