Journal: Free radical biology & medicine

Article Title: New insights in the molecular regulation of the NADPH oxidase 2 activity: Negative modulation by Poldip2.

doi: 10.1016/j.freeradbiomed.2023.02.019

Figure Lengend Snippet: Fig. 3. Comparison of gp91phox, Poldip2 and p22phox expression in monocytes, neutrophils and macrophages isolated from healthy donor blood. M0: monocyte; N: neutrophil; MA1 and MA2 macrophage type 1 (pro- inflammatory and anti-tumoral) and 2 (pro-tumoral and anti-inflammatory). 12 μg of total protein (cell lysates) are loaded. The ponceau staining of these western blots is shown in Fig. S5. All samples are from the same donor. Experiment was repeated independently twice with similar results for blood from two healthy donors.

Article Snippet: The membranes were incubated overnight at 4 ◦C with specific monoclonal or polyclonal antibodies: anti-gp91 (anti-gp91phox; 54.1; mouse monoclonal, dilution 1:1500; Santa Cruz), anti-p22 (44.1; mouse monoclonal; dilution 1:1500; Santa Cruz); anti-Poldip2 (ab181841; rabbit monoclonal; , dilution 1:1500; Abcam), anti-p67 (07-002; rabbit polyclonal; dilution 1:1000; Millipore); Anti-p47 (BD 610354; rabbit polyclonal; dilution 1:1500; Bioscience), anti-Rac1 (ARCO3; mouse monoclonal; dilution 1:500; Cytoskeleton), anti-NOX4 (ab109225; rabbit monoclonal; dilution 1:1500; Abcam).

Techniques: Comparison, Expressing, Isolation, Staining, Western Blot

Journal: Free radical biology & medicine

Article Title: New insights in the molecular regulation of the NADPH oxidase 2 activity: Negative modulation by Poldip2.

doi: 10.1016/j.freeradbiomed.2023.02.019

Figure Lengend Snippet: Fig. 5. Poldip2 interaction with purified membrane fraction isolated from human neutrophils. Poldip2 (145 μl; 0.2 μM) and neutrophil membrane fractions (MF; 1.75 μl; 1.28 μM), containing the cytb558 (gp91phox/p22phox), were pre-incubated for 1 h with the cytb558/Poldip2 ratio of 1:20 (mol:mol). The MF and Polidp2 (alone) were treated similarly. The three samples were centrifuged at 190 000×g for 1h30. Pellet and supernatant were separated and analyzed by Western blot using antibodies raised against Nox2 (1:1500), p22phox (1:1500) and Poldip2 (1:1500). Experiments were performed at least three times with similar results.

Article Snippet: The membranes were incubated overnight at 4 ◦C with specific monoclonal or polyclonal antibodies: anti-gp91 (anti-gp91phox; 54.1; mouse monoclonal, dilution 1:1500; Santa Cruz), anti-p22 (44.1; mouse monoclonal; dilution 1:1500; Santa Cruz); anti-Poldip2 (ab181841; rabbit monoclonal; , dilution 1:1500; Abcam), anti-p67 (07-002; rabbit polyclonal; dilution 1:1000; Millipore); Anti-p47 (BD 610354; rabbit polyclonal; dilution 1:1500; Bioscience), anti-Rac1 (ARCO3; mouse monoclonal; dilution 1:500; Cytoskeleton), anti-NOX4 (ab109225; rabbit monoclonal; dilution 1:1500; Abcam).

Techniques: Purification, Membrane, Isolation, Incubation, Western Blot

Journal: Aging

Article Title: Contribution of membrane raft redox signalling to visfatin-induced inflammasome activation and podocyte injury.

doi: 10.18632/aging.205243

Figure Lengend Snippet: Figure 1. Effects of visfatin on aggregation of gp91phox, p47phox and MR clusters, O2·− production in podocytes. (A) Representative confocal microscopic images of MR clusters (Green) and aggregation of gp91phox (Red) and MR clusters and p47phox (Red) in podocytes (original magnification, 400x). Only overlaid images were presented. Yellow spots in overlaid images indicate co-localization of the MR and gp91phox or p47 phox. (B) Summarized data showing the effects of Visfatin, MR disruptor, MCD and NADPH oxidase inhibitors DPI on MR- gp91phox or MR-p47clusters (n = 6). Values are means ± SEM, showing fold changes as compared with control. (C) Summarized data showing the effects of visfatin on O2·− production (n = 5 − 7). Values are means ± SEM, showing fold changes as compared with control. Abbreviations: Ctrl: Control; Vis: Visfatin; DPI: diphenyleneiodonium; MCD: methyl-β-cyclodextrin. *P < 0.05 vs. control; #P < 0.05 vs. visfatin.

Article Snippet: For the assessment of MR colocalization with gp91phox and p47phox, podocytes were incubated overnight with primary antibodies, including anti-gp91phox (1:200 dilution, Santa Cruz, USA, Cat # Sc35503) and anti-p47phox (1:200 dilution, Santa Cruz, USA, Cat # Sc376614) for mouse antibodies.

Techniques: Control

Journal: JCI Insight

Article Title: KLF11 protects against abdominal aortic aneurysm through inhibition of endothelial cell dysfunction

doi: 10.1172/jci.insight.141673

Figure Lengend Snippet: ( A and B ) The production of superoxide was determined in HAECs. HAECs were infected with Ad- lacZ or Ad- KLF11 , or Ad-sh lacZ or Ad-sh KLF11 (10 MOI). After 48 hours, they were treated with TNF-α (2 ng/mL) or AngII (1 μM) for 2 hours in the presence of superoxide detection solution (fluorescent probes). ( C – E ) HAECs were infected with Ad- GFP or Ad- KLF11 (10 MOI) or transfected with control siRNA (siControl) or KLF11 siRNA (si KLF11 , 20 μM). After 48 hours, they were stimulated with TNF-α (2 ng/mL) or AngII (1 μM) for 24 hours. ( C and D ) The mRNA levels of NOX1 , NOX2 , NOX4 , and NOX5 were determined by qPCR. ( E ) Western blot to determine the expression of NOX2 in HAECs. ( F ) HAECs were transfected with siControl, si KLF11 or si KLF11 + NOX2 siRNA (si NOX2 ) (20 μM). After 48 hours, they were treated with TNF-α (2 ng/mL) or AngII (1 μM) for 2 hours in the presence of superoxide detection solution. ( G and H ) Representative DHE staining and quantification of superoxide production in HAECs. HAECs were transfected with siControl, si KLF11 , or si KLF11 +si NOX2 . After 48 hours, they were pretreated with NOX2 inhibitor (NOX2i, GSK2795039, 1 μM) for 1 hour, then stimulated with TNF-α (2 ng/mL) or AngII (1 μM) for 2 hours, followed by DHE staining of superoxide (red) and immunofluorescence staining of VE-cadherin (green). Nuclei stained by DAPI are shown in blue. Scale bar: 20 μm. ( I ) HAECs were infected with Ad- lacZ or Ad-flag- KLF11 . After 48 hours, they were stimulated with TNF-α (2 ng/mL) for 4 hours, followed by ChIP assay using an antibody against flag or IgG. Data are presented as mean ± SEM. Two-way ANOVA followed by Holm-Sidak post hoc analysis ( A – D , F , H , and I ).

Article Snippet: NOX2 antibody (NBP2-67680) was purchased from Novus Biologicals.

Techniques: Infection, Transfection, Control, Western Blot, Expressing, Staining, Immunofluorescence

Journal: JCI Insight

Article Title: KLF11 protects against abdominal aortic aneurysm through inhibition of endothelial cell dysfunction

doi: 10.1172/jci.insight.141673

Figure Lengend Snippet: ( A – E ) Human aortic smooth muscle cells (HASMCs) were treated for 24 hours with the conditioned media from ECs (EC-CM) that had been transfected with siControl, si KLF11 , or si KLF11 +si NOX2 (20 μM), or infected with Ad- GFP or Ad- KLF11 (10 MOI) and subsequently stimulated for 1 hour with TNF-α (2 ng/mL) 48 hours after siRNA transfection or adenovirus infection and cultured in fresh opti-MEM for an additional 4 hours. ( A – D ) qPCR ( A and C ) and Western blot ( B and D ) to examine expression of SMC-specific contractile markers (smooth muscle α-actin [SMA], calponin, and smooth muscle 22–α [SM22α]), proinflammatory cytokines (MCP-1 and IL-6), and metalloproteinases (MMP2 and MMP9). ( E ) HASMCs were cultured in EC-CM for 48 hours, followed by immunostaining of TUNEL. Scale bar: 20 μm. ( F – H ) Schematics of the in vitro coculture system using a Transwell. HAECs (upper chamber) transfected with siControl, si KLF11 (20 μM), or si KLF11 +si NOX2 or infected with Ad- GFP or Ad- KLF11 (10 MOI) were cultured for 48 hours followed by TNF-α (2 ng/mL) stimulation for 1 hour separately from HASMCs, changed to fresh opti-MEM, and then cocultured with HASMCs (bottom) in fresh opti-MEM for 24 hours. The expression of BAX in HASMCs was assayed by Western blot ( G and H ). Data are mean ± SEM from 3 independent experiments. Two-way ANOVA followed by Holm-Sidak post hoc analysis ( A , C , E , G , and H ).

Article Snippet: NOX2 antibody (NBP2-67680) was purchased from Novus Biologicals.

Techniques: Transfection, Infection, Cell Culture, Western Blot, Expressing, Immunostaining, TUNEL Assay, In Vitro