transam nrf2 elisa-based transcription factor assay kit  (Active Motif)

Journal: Redox Biology

Article Title: Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia

doi: 10.1016/j.redox.2023.102901

Figure Lengend Snippet: Effects of 11 hit compounds on cell viability and NRF2 activity in H1299-YFP cells, and cell proliferation and cell death in NRF2 Mut ESCC cells (KYSE70 and KYSE180).

Article Snippet: NRF2-ARE binding assays were performed with an ELISA-based TransAM NRF2 Assay Kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions.

Techniques: Activity Assay, Inhibition

Journal: Redox Biology

Article Title: Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia

doi: 10.1016/j.redox.2023.102901

Figure Lengend Snippet: PYR inhibited NRF2 expression in the esophageal epithelium of Sox2CreER;LSL-Nrf2 E79Q/+ mice . (A) Experimental design to test the effect of PYR (30 mg/kg/day, p.o. ) on the NRF2 high phenotype in the mouse esophagus; (B) Expression of NRF2 and its target genes (GCLC, GCLM, PKM2) in the esophageal epithelium of Sox2CreER;LSL-Nrf2 E79Q/+ mice as detected with Western blotting; (C) Histology and expression of NRF2, NRF2 target genes, and BrdU in the esophageal epithelium of Sox2CreER;LSL-Nrf2 E79Q/+ mice as detected with IHC. Scale bar = 50 μm.

Article Snippet: NRF2-ARE binding assays were performed with an ELISA-based TransAM NRF2 Assay Kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions.

Techniques: Expressing, Western Blot

Journal: Redox Biology

Article Title: Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia

doi: 10.1016/j.redox.2023.102901

Figure Lengend Snippet: High-throughput screening for NRF2 inhibitors from two compound libraries (1 μM) using NQO1-YFP H1299 cells . (A) Flow chart of the screening strategy; (B) Time-dependent changes of the YFP signal (NRF2 activity); (C) Time-dependent changes of the mCherry signal (cell viability); Negative control: exposure to a known NRF2 activator (CDDO, 200 nM); Positive control: exposure to the vehicle. (D) Prestwick library (1280 FDA-approved drug compounds); (E) Asinex library (34,560 compounds). Hit compounds were selected if the CDDO-induced NRF2 activity was inhibited by >50% and cell survival was >80%. Three compounds from the Asinex library were defined as borderline hits because their inhibition of NRF2 activity was >50%, yet their effects on cell survival were ∼80%.

Article Snippet: NRF2-ARE binding assays were performed with an ELISA-based TransAM NRF2 Assay Kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions.

Techniques: High Throughput Screening Assay, Activity Assay, Negative Control, Positive Control, Inhibition

Journal: Redox Biology

Article Title: Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia

doi: 10.1016/j.redox.2023.102901

Figure Lengend Snippet: PYR and MIT downregulated NRF2 and NQO1 expression in NRF2 Mut -KYSE70 cells in a dose- and time-dependent manner. (A) Dose-dependent downregulation of the expression of nuclear NRF2 and cytoplasmic NQO1 by PYR; (B) Time-dependent downregulation of the expression of nuclear NRF2 and cytoplasmic NQO1 by PYR (10 μM); (C) Dose-dependent downregulation of NRF2 and NQO1 expression by MIT; (D) Time-dependent downregulation of NFR2 and NQO1 expression by MIT (10 nM). *P < 0.05, **P < 0.01.

Article Snippet: NRF2-ARE binding assays were performed with an ELISA-based TransAM NRF2 Assay Kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions.

Techniques: Expressing

Journal: Redox Biology

Article Title: Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia

doi: 10.1016/j.redox.2023.102901

Figure Lengend Snippet: PYR shortened NRF2 half-life and promoted NRF2 ubiquitination in NRF2 Mut ESCC cells. (A) NRF2 half-life was shortened by PYR (10 μM) and MIT (20 nM), but not by MTX (50 nM), in NRF2 Mut -KYSE70 cells. (B) NRF2 half-life was reduced by PYR (10 μM) and MIT (20 nM) in NRF2 Mut -KYSE180 cells. (C) NRF2 half-life was not changed by PYR (10 μM) and MIT (20 nM) in NRF2 WT -KYSE450 cells. (D) PYR (10 μM), but not MIT (20 nM), promoted NRF2 ubiquitination in NRF2 Mut -KYSE70 cells in the presence or absence of MG132 (a proteasomal inhibitor). (E) PYR (10 μM), but not MIT (20 nM), promoted NRF2 ubiquitination in NRF2 Mut -KYSE180 cells in the presence or absence of MG132. (F) PYR (10 μM), but not MIT (20 nM), slightly promoted NRF2 ubiquitination in NRF2 WT -KYSE450 cells only in the presence of MG132. *P < 0.05.

Article Snippet: NRF2-ARE binding assays were performed with an ELISA-based TransAM NRF2 Assay Kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions.

Techniques:

Journal: Redox Biology

Article Title: Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia

doi: 10.1016/j.redox.2023.102901

Figure Lengend Snippet: NRF2 high esophageal phenotype in Sox2CreER;LSL-Nrf2 E79Q/+ mice. (A) Schematic figure of the LSL-Nrf2 E79Q allele and experimental design. (B) Expression of NRF2, NRF2 target genes (GCLC and GCLM), and a keratinization marker (loricrin) in the esophageal epithelium of Sox2CreER;LSL-Nrf2 E79Q/+ mice as detected with Western blotting; (C) Histology of the esophageal epithelium at 4 and 6 weeks after tamoxifen activation (H&E) and expression of NRF2, loricrin, and a proliferation marker (BrdU) in the mouse esophageal epithelium as detected with IHC. (D) 18 F-FDG PET/CT of mouse esophagus before tamoxifen induction (Week 0) and after tamoxifen induction (Week 1). Transverse views of the esophagus (arrow, highlighted by the contrast agent) of one mouse (M313) are shown. ****P < 0.001.

Article Snippet: NRF2-ARE binding assays were performed with an ELISA-based TransAM NRF2 Assay Kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions.

Techniques: Expressing, Marker, Western Blot, Activation Assay, Positron Emission Tomography-Computed Tomography

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Uric Acid Protects against Focal Cerebral Ischemia/Reperfusion-Induced Oxidative Stress via Activating Nrf2 and Regulating Neurotrophic Factor Expression

doi: 10.1155/2018/6069150

Figure Lengend Snippet: Effect of UA on Nrf2 protein distribution, HO-1 protein expression, and Nrf2 DNA-binding activity in cerebral cortices in rats. (a, b) Representative western blot of nuclear Nrf2. Quantification of nuclear Nrf2 normalized with histone H3. (c, d) Representative western blot of cytosolic Nrf2 and HO-1. Quantification of cytosolic Nrf2 and HO-1 normalized with β -actin. (e) Nuclear Nrf2 DNA-binding activity. Data are means ± SE ( n = 4). ∗ P < 0.05, ∗∗ P < 0.01 vs. the vehicle-treated I/R group; # P < 0.05, ## P < 0.01 vs. the UA-treated I/R group.

Article Snippet: A commercially available Active Motif's (Carlsbad, CA, USA) ELISA-based TransAM® Nrf2 kit was used to analyze Nrf2 DNA-binding activity [ ].

Techniques: Expressing, Binding Assay, Activity Assay, Western Blot

Journal: Free radical biology & medicine

Article Title: Ammonia Promotes Endothelial Cell Survival via the Heme Oxygenase-1-Mediated Release of Carbon Monoxide

doi: 10.1016/j.freeradbiomed.2016.11.029

Figure Lengend Snippet: Ammonia stimulates HO-1 promoter activity via the Nrf2/ARE complex in HUVECs. (A) Ammonia-mediated HO-1 gene expression requires de novo RNA synthesis. Effect of actinomycin D (ActD; 0.10µg/ml) on NH4Cl (5.0mM for 8 hour)-mediated increase in HO-1 protein. (B) Ammonia stimulates HO-1 promoter activity. Cells were transfected with a HO-1 promoter construct (E1) or a mutated HO-1 promoter construct (M739) and a Renilla luciferase construct, treated with NH4Cl (5.0mM for 6 hours), and then analyzed for luciferase activity. In some instances, a dominant-negative Nrf2 (dnNrf2) construct was co-transfected into the cells. (C) Time-course of Nrf2 protein expression in native non-transfected cells after administration of NH4Cl (5.0mM). (D) Time-course of Nrf2 mRNA expression native non-transfected after administration of NH4Cl (5.0mM). (E) Ammonia stimulates Nrf2 activity. Native non-transfected cells were treated with NH4Cl (5.0mM) for 6 hours and nuclear extracts analyzed for Nrf2 binding by ELISA. O.D., optical density. (F) Effect of ammonia on GSK3β activity. Native non-transfected cells were treated with NH4Cl (5.0mM) for various times (0–24 hours) or with the phosphatidylinositol-3-kinase inhibitor LY2942002 (LY;10µM) for 4 hours. Protein and mRNA expression was quantified by scanning laser densitometry and normalized with respect to β-actin or GSK3β and 18 S rRNA, respectively, and expressed relative to that of control, untreated cells (open bars). Results are means ± SEM (n=3–5). *Statistically significant effect of NH4Cl or LY.

Article Snippet: The supernatant was collected and Nrf2 activity determined by measuring the binding of Nrf2 to the ARE using the ELISA-based TransAM Nrf2 kit (Active Motif, Carlsbad, CA).

Techniques: Activity Assay, Expressing, Transfection, Construct, Luciferase, Dominant Negative Mutation, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Free radical biology & medicine

Article Title: Ammonia Promotes Endothelial Cell Survival via the Heme Oxygenase-1-Mediated Release of Carbon Monoxide

doi: 10.1016/j.freeradbiomed.2016.11.029

Figure Lengend Snippet: Ammonia-induced HO-1 expression is dependent on oxidative stress in HUVECs. (A) Effect of N-acetyl-L-cysteine (NAC; 10mM) or rotenone (Rot;5µM) on NH4Cl (5.0mM for 8 hours)-mediated intracellular ROS production. (B) Effect of NAC (10mM) on NH4Cl (5.0mM for 24 hours)-mediated HO-1 protein expression. (C) Effect of NAC (10mM) on NH4Cl (5.0mM for 6 hours)-mediated Nrf2 activation. O.D., optical density. (D) Effect of Rot (5µM) on NH4Cl (5.0mM for 24 hours)-mediated HO-1 protein expression. (E) Effect of apocynin (Apo;300µM) on NH4Cl (5.0mM for 24 hours)-mediated HO-1 protein expression. (F) Effect of allopurinol (Allo;100µM) on NH4Cl (5.0mM for 24 hours)-mediated HO-1 protein expression. HO-1 protein was quantified by scanning laser densitometry and normalized with respect to β-actin, and expressed relative to that of control, untreated cells (open bars). Results are means ± SEM (n=3–6). *Statistically significant effect of NH4Cl.

Article Snippet: The supernatant was collected and Nrf2 activity determined by measuring the binding of Nrf2 to the ARE using the ELISA-based TransAM Nrf2 kit (Active Motif, Carlsbad, CA).

Techniques: Expressing, Activation Assay