| Assay Method Information | |
| | NOX3 Inhibition Assay |
| Description: | Evaluation of the NOX4 inhibitors on NOX3 activity is assessed in HEK293 cells stably overexpressing human NOX3 (hNOX3).Cells are cultured in the DMEM media containing 4.5 g/L glucose supplemented with 10% fetal calf serum (FCS), Puromycin 1 μg/mL and Blasticidin 1.5 μg/mL in an incubator at 37° C. with 5% CO2. Cells are seeded in 384 well plates for 2 days. For the assay cells are washed and treated with several concentrations (10 nM-30 μM) of test compound (diluted in DMSO) or 100 μM DPI (as a positive control in assay buffer consisting of PBS), and incubated for 2 hours at 24° C. in a humidified incubator. Cells are then stimulated with PMA at 0.1 μM to induce NOX3-dependent ROS production. Finally, L-012 is added at 100 μM and incubated for 2 hours at 24° C. in a humidified incubator. Luminescence is measured with an PHERAstar multimode reader. |
| Affinity data for this assay | |
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