| Assay Method Information | |
| | NOX1 Inhibition Assay |
| Description: | Evaluation of the NOX4 inhibitors on NOX1 activity is assessed in HEK293 cells stably overexpressing human NOX1 (hNOX1).Cells are cultured in the DMEM media containing 4.5 g/L glucose supplemented with 10% fetal calf serum (FCS), Puromycin 1 μg/mL, Blasticidin 1.5 μg/mL, Geneticin (G418) 200 μg/mL and Hygromycin 100 μg/mL in an incubator at 37° C. with 5% CO2. For the assay cells are seeded in 384 well plates in DMEM media with 10% FCS. After 24 h, cells are washed, treated for 30 mins with several concentrations (30 nM-100 μM) of test compounds and then stimulated with phorbol myristate acetate (PMA) at 1 μM to induce NOX1-dependent ROS production (except wells used as a positive control). Lastly, 8-amino-5-chloro-2,3-dihydro-7-phenyl-pyrido[3,4-d]pyridazine-1,4-dione sodium salt (L-012, CAS 143556-24-5) is added at 400 μM to all wells, and cells are incubated in an incubator for an additional 3 h. Afterwards luminescence is measured with a SpectraMax Paradigm Microplate Reader. Data can be found in table 2. |
| Affinity data for this assay | |
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