elisa-based transam kits  (Active Motif)

Journal: Mediators of Inflammation

Article Title: The Role of Ca 2+ /PI3K/Akt/eNOS/NO Pathway in Astragaloside IV–Induced Inhibition of Endothelial Inflammation Triggered by Angiotensin II

doi: 10.1155/2024/3193950

Figure Lengend Snippet: Effect of astragaloside IV (AS-IV) on angiotensin II (Ang II)-induced increases in the protein levels of phosphorylated IκBα (A) and activities of NF-κB (B) in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were preincubated with AS-IV (30 µmol/L) or sodium nitroprusside (SNP, 30 µmol/L) in the presence or absence of N G -monomethyl-L-arginine (L-NMMA, 1 mmol/L), LY294002 (10 µmol/L), or ethylene glycol tetraacetic acid (EGTA, 500 nmol/L) for 1 h and then incubated with Ang II (1 µmol/L) for an additional 30 min. The protein levels of phosphorylated IκBα and activities of NF-κB in HUVECs were measured using a cell-based enzyme-linked immunosorbent assay (ELISA) and a specific TransAM NF-κB p65 Transcription Factor Assay Kit, respectively. ⁣ ∗ p < 0.05 compared with the control group; † p < 0.05 compared with HUVECs exposed to Ang II in the presence of AS-IV.

Article Snippet: The NF-κB activity was determined using a nonradioactive, ELISA-based TransAM NF-κB p65 transcription factor assay kit (Active Motif, Carlsbad, CA, USA) as we described previously [ ].

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Transcription Factor Assay, Control

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: Frontiers in Immunology

Article Title: MSC therapy ameliorates experimental gouty arthritis hinting an early COX-2 induction

doi: 10.3389/fimmu.2023.1193179

Figure Lengend Snippet: Regulation of inflammasome components and NF-kB pathway by Ad-MSC in the synovium of MSU-injected rabbits. (A) Protein expression of inflammasome components is modulated in arthritic rabbits treated with Ad-MSC. Activated NLRP3, pro-caspase-1, IL-1β, and IL-18 were assessed by western blot. EZ blue staining was used as protein loading control. Results are normalized by EZ blue staining and expressed as a fold-change of the healthy group. Bars show the mean and SEM. (B) Representative images and quantification of NLRP3 antigen staining in the synovium of Control, MSU and MSU+MSC groups. Bars show the mean and SEM (C) NF-kB p65 was analyzed by TransAM kit assay. Results are expressed in optical density (OD) units. Bars show the mean and SEM. IL, interleukin; NLRP3, NLR Family Pyrin Domain Containing 3; MSC, mesenchymal stem cells; MSU, monosodium urate.

Article Snippet: NF-κB activation was assessed using an ELISA-based TransAM NF-κB p65 kit (Active Motif, CA, USA) in accordance with manufacturer’s protocol.

Techniques: Injection, Expressing, Western Blot, Staining