High Nrf2 protein in human AML cells is because of elevated Nrf2 RNA levels. (A) Nuclear extracts were obtained from human AML samples and control cells, extracts were separated by SDS-PAGE, and Western blot analysis was conducted for Nrf2 and Keap1 protein levels. Nuclear blots were reprobed with TATA binding protein (TBP) to confirm sample loading and cytosolic blots with GAPDH. (B) AML cells and control cells washed with PBS and incubation with 10μM H₂DCFDA for 15 minutes. Cells were then assessed for H₂DCFDA oxidation by the use of flow cytometry. (C) THP-1 cells were treated with NAC for up to 8 hours. Nuclear extracts were obtained and analyzed for Nrf2 expression. Nuclear blots were reprobed with TBP to confirm equal sample loading. The numbers under the Western blot indicate ROS levels in NAC-treated THP-1 cells. (D) RNA was extracted from AML cells and control cells and Nrf2 and Keap1 mRNA was measured with real-time PCR. mRNA expression was normalized to GAPDH mRNA levels. *Statistical significance (P < .05) between Nrf2 and Keap1 mRNA levels where indicated.