anti p22 phox  (Santa Cruz Biotechnology)

Journal: Biomedicines

Article Title: p75NTR Modulation Reduces Oxidative Stress and the Expression of Pro-Inflammatory Mediators in a Cell Model of Rett Syndrome

doi: 10.3390/biomedicines12112624

Figure Lengend Snippet: Impact of LM11A-31 on the expression of NADPH oxidase 4 subunits. Immunofluorescence and quantitative analysis of ( A ) NOX4 and ( B ) p22 phox fluorescence intensity in control fibroblast (HC-1), Rett syndrome fibroblasts (RTT-1), and RTT fibroblasts treated with LM11A-31 (0.1 μM for 24 h) (RTT-1+LM). Cells were fixed in 4% PFA and stained with antibodies against NOX4 (red) and p22 phox . DAPI was employed for nuclear counterstaining. n = 5–6 biological replicates. Images were acquired using the Leica TCS SP8 confocal microscope and Leica Application Suite X (LAS X) software at 40× magnification. Scale bar: 50 µm. Data are expressed as mean ± SD. Statistical analysis was performed using a one-way ANOVA, followed by Tukey’s post hoc test. Statistical significance is indicated as follows: *** p < 0.001. “a” indicates statistical significance vs. HC-1; “b” indicates statistical significance vs. RTT-1.

Article Snippet: The following primary antibodies were used in the immunocytochemistry experiments: anti-NGF (Santa Cruz Biotechnology, Dallas, TX, USA, sc-365944), anti-p75NTR (Santa Cruz Biotechnology, Dallas, TX, USA, sc-271708), anti-8-OHdG (Santa Cruz Biotechnology, Dallas, TX, USA, sc-66036), anti-4-HNE (Abcam, Cambridge, UK, ab46545), anti-GSH (Abcam, Cambridge, UK, ab19534), anti-NOX4 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-518092), anti-p22 phox (Santa Cruz Biotechnology, Dallas, TX, USA, sc-130551), anti-IL-6 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-129128), anti-IL-8 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-8427), anti-PPAR-α (Abcam, Cambridge, UK, ab314112), anti-PPAR-β/δ (Abcam, Cambridge, UK, ab23673), anti-PPAR-γ (Santa Cruz Biotechnology, Dallas, TX, USA, sc-7273), anti-PGC1α (Santa Cruz Biotechnology, Dallas, TX, USA, sc-13067), anti-Sirt1(Santa Cruz Biotechnology, Dallas, TX, USA, sc-74465), and anti-Nrf2 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-365949).

Techniques: Expressing, Immunofluorescence, Fluorescence, Control, Staining, Microscopy, Software

Journal: Journal of Translational Medicine

Article Title: LRRC8A drives NADPH oxidase-mediated mitochondrial dysfunction and inflammation in allergic rhinitis

doi: 10.1186/s12967-024-05853-w

Figure Lengend Snippet: LRRC8A mRNA expression correlates with NADPH oxidase subunits and clinical characteristics, with LRRC8A, NOX1, NOX4, and p22 phox upregulated in the nasal mucosa of allergic rhinitis patients compared to controls. A–G qRT-PCR was used to detect the mRNA expression levels of LRRC8A, NOX1, NOX4, p22 phox , IL-4, IL-5, and IL-13 in human nasal mucosa (Con n = 18, AR n = 30). H–O Correlation analysis was conducted between LRRC8A mRNA and the mRNA levels of NOX1, NOX4, p22 phox , IL-4, IL-5, IL-13(Con n = 18, AR n = 30), serum total IgE(Con n = 15, AR n = 26), and serum Phadiatop Test-specific IgE (Con n = 15, AR n = 24). P Protein expression of LRRC8A, NOX1, NOX4, and p22 phox in human nasal mucosal homogenates as detected by Western blotting (Con n = 5, AR n = 5). Q Immunohistochemical analysis showing the expression of LRRC8A, NOX1, and NOX4 in the epithelial layer(scale bar: 20 μm). The circled regions highlight the areas where these proteins are expressed (Con n = 8, AR n = 8). Data are presented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: The proteins were then transferred to a PVDF membrane and blocked with 5% non-fat milk for 1.5 h. The membranes were incubated overnight at 4 °C with primary antibodies against NOX1 (1:1000, #17772-1-AP, proteintech), NOX4 (1:4000), p22 phox (1:1000, #sc-271968, Santa Cruz Biotechnology), LRRC8A (1:2000, #ab254389, Abcam), NF-κB p65, and phospho-NF-κB p65 (1:1000, #8242, #3033, Cell Signaling Technology).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemical staining

Journal: Journal of Translational Medicine

Article Title: LRRC8A drives NADPH oxidase-mediated mitochondrial dysfunction and inflammation in allergic rhinitis

doi: 10.1186/s12967-024-05853-w

Figure Lengend Snippet: LRRC8A regulates the expression of NADPH oxidase subunits and ROS production. A, E Western blotting analysis of LRRC8A protein expression in HNEpCs after stimulation with IL-13 for 12 h. B, F, G Validation of siLRRC8A knockdown efficiency at the mRNA and protein levels. C, M Measurement of Cl − expression in HNEPCs using MQAE (scale bar = 50 μm). D, N After being stimulated with IL-13, HNEPCs transfected with siLRRC8A and NCsirna were measured for ROS expression using DCFH-DA (scale bar = 50 μm). H–L Western blotting analysis of LRRC8A, NOX1, NOX4, and p22 phox protein expression in HNEPCs. * P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: The proteins were then transferred to a PVDF membrane and blocked with 5% non-fat milk for 1.5 h. The membranes were incubated overnight at 4 °C with primary antibodies against NOX1 (1:1000, #17772-1-AP, proteintech), NOX4 (1:4000), p22 phox (1:1000, #sc-271968, Santa Cruz Biotechnology), LRRC8A (1:2000, #ab254389, Abcam), NF-κB p65, and phospho-NF-κB p65 (1:1000, #8242, #3033, Cell Signaling Technology).

Techniques: Expressing, Western Blot, Knockdown, Transfection

Journal: Journal of Translational Medicine

Article Title: LRRC8A drives NADPH oxidase-mediated mitochondrial dysfunction and inflammation in allergic rhinitis

doi: 10.1186/s12967-024-05853-w

Figure Lengend Snippet: Knockdown of LRRC8A suppresses NOX1, NOX4, and p22 phox -mediated mitochondrial dysfunction and NF-κB pathway activation in the AR mouse model. A–D Protein expression levels of NOX1, NOX4, and p22 phox were assessed by Western blotting analysis. E, F ROS and mtROS levels were measured using DCFH-DA and Mitosox fluorescent probes, respectively. (scale bar: 50 μm). G Mn-SOD enzyme activity levels were measured among different groups. H, I Protein expression levels of NF-κB pathway-related proteins, including p65 and P-p65, were determined by Western blotting analysis in the AR mouse. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: The proteins were then transferred to a PVDF membrane and blocked with 5% non-fat milk for 1.5 h. The membranes were incubated overnight at 4 °C with primary antibodies against NOX1 (1:1000, #17772-1-AP, proteintech), NOX4 (1:4000), p22 phox (1:1000, #sc-271968, Santa Cruz Biotechnology), LRRC8A (1:2000, #ab254389, Abcam), NF-κB p65, and phospho-NF-κB p65 (1:1000, #8242, #3033, Cell Signaling Technology).

Techniques: Knockdown, Activation Assay, Expressing, Western Blot, Activity Assay

Journal: Journal of Translational Medicine

Article Title: LRRC8A drives NADPH oxidase-mediated mitochondrial dysfunction and inflammation in allergic rhinitis

doi: 10.1186/s12967-024-05853-w

Figure Lengend Snippet: LRRC8A-mediated mechanism of nasal mucosal epithelial cell inflammation: LRRC8A activation by allergen inhalation increases Th2 cytokine secretion, leading to VRAC-associated LRRC8A upregulation. This induces up-regulation of NOX1, NOX4 and p22 phox , forming NOX1/NOX4 and p22 phox complexes, causing overproduction of ROS, impairing Mn-SOD activity, TMRM, and mtDNA copy number, leading to mitochondrial dysfunction in epithelial cells. At the same time, the NF-κB pathway was activated, which ultimately promoted nasal mucosal epithelial inflammation

Article Snippet: The proteins were then transferred to a PVDF membrane and blocked with 5% non-fat milk for 1.5 h. The membranes were incubated overnight at 4 °C with primary antibodies against NOX1 (1:1000, #17772-1-AP, proteintech), NOX4 (1:4000), p22 phox (1:1000, #sc-271968, Santa Cruz Biotechnology), LRRC8A (1:2000, #ab254389, Abcam), NF-κB p65, and phospho-NF-κB p65 (1:1000, #8242, #3033, Cell Signaling Technology).

Techniques: Activation Assay, Activity Assay

Journal: Antioxidants

Article Title: Sirtuin1 Mediates the Protective Effects of Echinacoside against Sepsis-Induced Acute Lung Injury via Regulating the NOX4-Nrf2 Axis

doi: 10.3390/antiox12111925

Figure Lengend Snippet: Activated SIRT1 inhibited NOX4 activation and promoted NOX4 ubiquitination degradation. ( A ) Endothelial cells were treated with different concentrations of ECH and SIRT1 activator resveratrol. Immunofluorescence was used to assess the co-staining of NOX4 (red) and p22 phox (green). Bars represent 20 μm. The microscope’s magnifications are the same. ( B ) CO-IP was performed to assess the binding of NOX4 and p22 phox. ( C ) CO-IP was performed to assess the binding of SIRT1 and p22 phox. ( D ) CO-IP was used to measure the ubiquitination level of NOX4. ( E ) CO-IP was used to measure the acetylation level of Nrf2. ( F ) Under Control siRNA and SIRT1 siRNA treatments, immunofluorescence was used to assess the co-staining of NOX4 (red) and p22 phox (green). Bars represent 20 μm. The microscope’s magnifications are the same. ( G ) Under Control siRNA and SIRT1 siRNA treatments, CO-IP was performed to assess the binding of NOX4 and p22 phox. ( H ) Under Control siRNA and SIRT1 siRNA treatments, CO-IP was performed to assess the binding of SIRT1 and p22 phox. ( I ) Under Control siRNA and SIRT1 siRNA treatments, CO-IP was used to measure the ubiquitination level of NOX4. The data are presented as mean ± standard deviation, and all experiments were repeated independently at least three times.

Article Snippet: The membranes were then incubated with specific primary antibodies overnight at 4 °C, including β-actin antibody (1:5000, Cat# 66009-1-Ig, Proteintech, China), VCAM-1 antibody (1:1000, Cat# bs-0920R, Bioss, Boston, MA, USA), ICAM-1 antibody (1:1000, Cat# 10020-1-AP, Proteintech, China), SIRT1 polyclonal antibody (1:1000, CAT# 13161-1-AP, Proteintech, China), HO-1 antibody (1:2000, Cat# ab189491, Abcam, Cambridge, UK), NQO-1 antibody (1:2000, CAT# ab80588, Abcam, UK), NOX4 antibody (1:1000, Cat# 14347-1-AP, Proteintech, China), p65 antibody (1:1000, Cat# 8242T, CST, Danvers, MA, USA), Pho-p65 antibody (1:1000, Cat# 3033T, CST, USA), IκB α antibody (1:1000, Cat# ab32518, Abcam, UK), Pho-IκB α antibody (1:10,000, Cat# ab133462, Abcam, UK), Pho-JNK antibody (1:1000, Cat# 9251S, CST, USA), JNK antibody (1:1000, Cat# 9252T, CST, USA), ERK1/2 antibody (1:1000, Cat# 4695T, CST, USA), Pho-ERK1/2 antibody (1:1000, Cat# 4370T, CST, USA), Pho-p38 antibody (1:1000, Cat# 28796-1-AP, Proteintech, China), p38 antibody (1:1000, Cat# 14064-1-AP, Proteintech, China), Nrf2 antibody (1:1000, Cat# 16396-1-AP, Proteintech, China), Ubquination antibody (1:500, Cat# A162, Abclonal, Wuhan, China) and p22 phox antibody (1:1000, Cat# A10694, ABclonal, China).

Techniques: Activation Assay, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Binding Assay, Standard Deviation