p45 competitively inhibits Nrf2-mediated transcriptional activation. (A) Quantitative RT-PCR of platelet genes and cytoprotective genes in p45-null megakaryocytes. The relative values were calculated against the values of WT megakaryocytes. (B) Immunoblot analysis of nuclear extracts of p45-null and WT megakaryocytes cultured from fetal livers. Anti-Nrf2 and anti–lamin B antibodies were used. (C) Comparison of protein abundance of p45 and Nrf2. FLAG-tagged p45 and FLAG-tagged Nrf2 were transiently overexpressed in 293T cells. Whole-cell extracts were prepared and subjected to immunoblot analysis with anti-FLAG or anti–lamin B antibodies. and ◀ indicate FLAG-Nrf2 and FLAG-p45, respectively. (D) Comparison of transcriptional activation abilities of p45 and Nrf2. Equal amount of the expression vectors were introduced into 293T cells with a luciferase gene driven by MARE of the Nqo1 promoter in triplicate as a reporter gene. (E) Reporter assay in 293T cells with the same reporter gene used in panel D. Expression vectors of Nrf2 and/or p45 without tags were added as effector molecules. (F) Comparison of transcriptional activation abilities of FLAG-tagged Nrf2 and FLAG-tagged fusion protein of the N-terminal half of Nrf2 and C-terminal half of p45 with the same reporter gene used in panel D. The protein expression detected by anti-FLAG antibody is shown below. The relative luciferase activities were calculated against the activity generated by the reporter gene alone (D-F). The average values of triplicate experiments are presented, and the error bars represent SD (A,D-F). The Student t test was used to calculate statistical significance. *P < .05; **P < .005 (A,D-F).