FIG. 1.

Nrf2 is acetylated by p300/CBP in vivo and in vitro. (A) Endogenous Nrf2 is acetylated. HCT116 cells were lysed under denaturing conditions. The cell lysates were diluted and subjected to immunoprecipitation (IP) by normal IgG or anti-Nrf2 (α-Nrf2) antibodies, followed by immunoblotting (IB) with antibodies specific for acetylated lysine (AcK). Tub, tubulin. (B) Sodium arsenite enhances acetylation of Nrf2. HCT116 cells were treated with 20 μm As(III) for the indicated times. Nrf2 acetylation levels were measured as described for panel A. (C) Nrf2 is acetylated by p300/CBP, not P/CAF. HEK293T cells were cotransfected with vectors expressing HA-tagged Nrf2 and the indicated HATs. The cell lysates were immunoprecipitated with anti-HA antibodies, followed by immunoblotting with antibodies specific for acetylated lysine. (D) Nrf2 is a bona fide substrate of p300. (Top) Purified GST-tagged Nrf2 proteins were incubated in the presence of ¹⁴C-labeled acetyl-CoA with immunoprecipitated p300 proteins from COS-1 cells overexpressing Flag-p300. The reaction mixtures were resolved on SDS-PAGE, followed by autoradiography. (Bottom) The same amounts of GST-Nrf2 proteins as used in the in vitro acetylation assay described above were subjected to Coomassie staining.