Fig 2: NRF2 promotes activation of the ATR–CHK1–CDC2 signaling pathway via its AAD-like domain and interacts with ATR at DNA damage sites. (A) Schematic illustration of the HA-tagged NRF2 mutants. (B) Western blotting of HA, p-ATR, and p-CHK1 in HEK293T cells treated with 60 nM CPT for 6 h and released for 2 h. (C) Western blotting of HA, p-ATR and p-CHK1 in HEK293T cells pretreated with 5 mM NAC for 1 h and exposed to 8 Gy Cs137 ?-rays. (D) Immunofluorescence analysis of NRF2 (red), ?H2AX (green), and DAPI (blue) in A549 cells exposed to 8 Gy Cs137 ?-rays. Scale bars, 20 µm. (E) Immunofluorescence analysis of NRF2 (red), ATR (green), and DAPI (blue) in A549 cells exposed to 8 Gy Cs137 ?-rays. Scale bars, 20 µm. (F) The co-localization of red (NRF2) and green (ATR) foci was analyzed by Image-Pro Plus 6.0. (G) Extracts of A549 cells exposed to 0 or 8 Gy Cs137 ?-rays were subjected to immunoprecipitation with an anti-ATR antibody or control IgG. Samples were immunoblotted with the indicated antibodies. (H) Extracts of A549 cells exposed to 0 or 8 Gy Cs137 ?-rays were subjected to immunoprecipitation with an anti-NRF2 antibody or control IgG. Samples were immunoblotted with the indicated antibodies. (I) Extracts of A549 cells treated with or without 30 nM CPT were subjected to immunoprecipitation with an anti-NRF2 antibody or control IgG. Samples were immunoblotted with the indicated antibodies. (J) GST pull-down assay following incubation of (1–338), (1–338) lacking amino acids 180–230 (?180–230), (1–338) in which the W amino acid at position 188 was replaced by A (W188A) or GST produced in E. coli with Flag-ATR produced in HEK293T cells.