Figure 4.

hMOF acetylates Nrf2 and promotes its nuclear retention. (A) hMOF acetylates Nrf2. Plasmids expressing HA-Nrf2 and Flag-hMOF were transfected respectively or together into 293T-cells for 48 h. Cell lysates were subjected to IP and IB analysis with indicated antibodies. (B) H₂O₂ promotes the acetylation of Nrf2 by hMOF. 293T-cells were transfected with plasmids expressing HA-Nrf2 with/without Flag-hMOF. Thirty-six hours later, the cells were left untreated or were treated with 100 μM H₂O₂ for additional 12 h. Then, cell lysates were subjected to IP and IB analysis with indicated antibodies. (C–D) hMOF acetylates Nrf2 at lysine 588. (C) 293T-cells were transfected with the plasmids expressing the indicated proteins. Forty-eight hours post-transfection, cells lysates were subjected to IP and IB analysis with indicated antibodies. (D) Recombinant wide-type Nrf2 and Nrf2K588R proteins were expressed in Escherichia coli, and the purified proteins were subjected to in vitro acetylation with/without recombinant hMOF. The products were subjected to IB analysis with indicated antibodies. (E) hMOF promotes Nrf2 nuclears retention. 293T-cells were transfected with control siRNA (NC-siRNA) or siRNA targeting hMOF (hMOF-siRNA) for 36 h. Then the cells were treated with or without 100 μM H₂O₂ for 12 h. The nuclear and cytoplasmic proteins were subjected to IB analysis with the indicated antibodies.