Figure 3.

TRL reacts with a thiol group in KEAP1 to activate Nrf2. (A) TRL (200 μM) in acetonitrile/PBS (1/1, v/v) was incubated with or without 20 mM N-acetylcysteine (NAC) for 10 m. The reaction mixtures were analyzed by thin-layer chromatography. (B) The reaction mixture was subjected to mass spectrometry. The molecular weight peak (m/z, in negative mode) corresponding to the NAC-TRL adducts was detected. (C) Cell lysates were treated with 100 μM of either TRL or rTRL for 30 m, followed by incubation with a thiol-reactive biotin BT-MA (1 μM) at 30 °C for 2 h. The beads were washed five times. Laemmli sample buffer was added, and the mixture was boiled for 5 m prior to western blot analysis. The blots were probed with an anti-KEAP1 antibody. (D) HCT116 cells were pretreated with MG132 (10 μM) for 2 h and then lysed with lysis buffer. The cell lysates were treated with TRL (100 μM) at 30 °C for 2 h, followed by the addition of anti-KEAP1 antibody at 4 °C for 2 h. Protein A/G agarose beads were added to the lysates at 4 °C for 2 h. The beads were washed five times. Laemmli sample buffer was added, and the mixture was boiled for 5 min. Nrf2 and KEAP1 proteins in the immunocomplex were monitored by western blotting. NM, not measurable.