FIGURE 5.

CRIF1-driven NRF2 ubiquitination and degradation are redox-independent. Total lysates of MCF-7 cells co-transfected with the indicated expression vectors for 24 h were treated with either 100 μm of t-BHQ or 5 μm of SFN for 6 h and were immunoprecipitated with an anti-His antibody (for NRF2) followed by analysis on WBs with anti-HA antibody (for ubiquitin). One-tenth of the total cell lysates was used to determine the expression level of each transfected DNA plasmid by WB analysis. Anti-V5 antibody was used to detect exogenously expressed NRF2, and the anti-FLAG antibody was used to detect either CRIF1 or KEAP1. The two proteins, FLAG-CRIF1 and FLAG-KEAP1, were identified by their molecular weights. FLAG-CRIF1 and FLAG-KEAP1 were detected at ∼30 and 70 kDa, respectively.