We measured NRF2 transcription activity by performing Luciferase reporter gene assays: 6 × 104 A549 cells were seeded in 24-well plates. Next day cells were co-transfected with 500 ng of pGL3-8xARE (NRF2 activity) or pGLHIF1.3 (HIF activity), together with 100 ng pGL4.74 (renilla luciferase). If required, cells were co-transfected with 500 ng of GFP-NRF2, GFP-NRF2-S40A or various amounts of Flag-Keap1. Transfections were performed using ViaFect (Promega, Madison, U.S.) following the manufacturer’s instructions. Seventy-two hours later, cell lysis was done by using passive lysing buffer (Promega, Madison, U.S.). Firefly and renilla luciferase were measured using the GloMax detection system (Promega) and normalized to renilla values.

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