FIG. 4.

Functional redundancy among different acetylation sites. (A) Arginine substitution for single or several adjacent lysine residue(s) does not change overall Nrf2 acetylation levels. Acetylation on Nrf2 was analyzed as described for HEK293T cells expressing the indicated HA-Nrf2 and p300. IP, immunoprecipitation; IB, immuno-blotting; α, anti. (B) HEK293T cells were cotransfected with vectors for the NQO1-ARE-dependent firefly luciferase reporter gene, TK Renilla luciferase gene, the indicated HA-Nrf2, and p300. Luciferase reporter gene activities were analyzed using the Promega dual-luciferase reporter gene assay system. Relative luciferase activities and standard deviations were calculated from three independent experiments. Inr, initiator. (C) Total cell lysates from the luciferase assay in panel B were subjected to immunoblot analysis with anti-HA antibodies. Tub, tubulin.