Fig. 5. Bixin activates the NRF2 signaling pathway by decreasing NRF2 ubiquitination and increasing NRF2 protein stability. (A) THP-1 cells were cotransfected with plasmids encoding the indicated proteins for 24 h. The transfected cells were treated with SF (5 μM), tBHQ (40 μM) or bixin (40 μM) along with MG132 (10 μM) for 4 h. Anti-NRF2 immunoprecipitates were analyzed by immunoblotting with anti-HA antibody for the detection of ubiquitin-conjugated NRF2. (B) THP-1 cells were treated with bixin (40 μM) along with MG132 (10 μM) for 4 h. Anti-NRF2 immunoprecipitates were analyzed by immunoblotting with ubiquitin antibody for the detection of endogenous unbiquitin-conjugated NRF2. (C) THP-1 cells were either left untreated or treated with bixin (40 μM) for 4 h. Cycloheximide (CHX, 50 μM) was added and the cells were lysed at the indicated time points. Cell lysates were subjected to immunoblot analysis using anti-NRF2 and anti-GAPDH antibodies. The intensities of the bands were quantified using the GeneTools software and plotted against time after CHX treatment to obtain the half-life values.