Immunoprecipitation and western blot analysis

Cells were lysed and immunoprecipitated with anti-NRF2 antibody (Cell Signaling Technology). 30 µL protein A/G agarose beads (GE Healthcare, Pittsburgh, PA, USA) were used for 4 h to detect the association between NRF2 and Keap1.Isolated protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene-fluoride membranes (Millipore, Billerica, MA, USA). The primary antibodies included HO1 (Abcam, 1/1,000, ab68477), NQO1 (Abcam, 1/1,000, ab28947), NRF2 (Novus, 1/1,000, NBP1-32822), FPRL1 (Novus, 1/1,000, NLS1878) and Keap1 (Santa Cruz Biotechnology, 1/200, sc-514914). Secondary antibodies (ZSGB-BIO, 1/3,000, ZB-2301 and ZSGB-BIO, 1/3,000, ZB-2305) were also employed. Equal protein loading among the samples was confirmed by the β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA) and the Lamin B1 antibody (Abcam, Cambridge, MA, USA). An enhanced chemiluminescence system (West Pico Kit, Pierce, Loughborough, UK) was employed to detect the membrane signals. The relative intensities were analyzed with Image J software.