Fig. 4

(A) HEK293T cells expressing Myc-Keap1 or its indicated C3IR mutants together with HA-ubiquitin (Ub) and FLAG-Nrf2 were treated with 10 μM MG132 for 6 hours, lysed, and subjected to immunoprecipitation with antibodies to FLAG. Asterisks indicate nonubiquitylated forms of FLAG-Nrf2. (B) Median fluorescence intensity (MFI) determined by flow cytometry for EGFP-Nrf2 expressed in HEK293T cells together with FLAG-Keap1 or its C3IR mutants. Data are means; n = 4. ****P < 0.0001 versus WT. (C) MFI of EGFP-Nrf2 expressed together with FLAG-Keap1 or its C3IR mutants that were treated with 1 μM MLN4924 for 24 hours before washout of MLN4924 and incubation of the cells with cycloheximide (CHX; 50 μg/ml) for the indicated times. Data are expressed relative to the value for time 0. (D to F) MFI of EGFP-Nrf2 expressed together with FLAG-Keap1 or its C3IR mutants during exposure to 25 μM tBHQ (D), 100 μM genistein (E), or 400 nM doxorubicin (F). (G) WT or Keap1 C3IR mutation knock-in HEK293T cells were exposed to 25 μM tBHQ, lysed, and subjected to immunoblot analysis with antibodies to Nrf2 (left). The relative abundance of endogenous Nrf2 was also quantitated (right). Data are means ± SEM; n = 5. (H and I) RT and real-time PCR analysis of HO1 (H) and NQO1 (I) mRNA abundance in WT or Keap1 C3IR mutation knock-in cells at 8 hours after treatment with 25 μM tBHQ. Data are means; n = 5. NS, not significant. (J and K) Flow cytometric analysis of WT or Keap1 C3IR mutation knock-in cells that were negative for annexin V and propidium iodide (PI) staining after treatment with 0.003% H₂O₂ (J) or 5 μM doxorubicin (K) for 24 hours. Data are means; n = 5.