Figure 6. The C-terminal 154KRR in p21 was Essential for the Binding and Upregulation of Nrf2.
(A) mKRR in p21 interacted with Nrf2 and was required for upregulation of Nrf2. Three p21 mutants were made. mRKRR, mKRR, and mKRK are mutants with RKRR, KRR, or KRK replaced with methionine residues, respectively. COS-1 cells were cotransfected with expression vectors for Nrf2 and the indicated p21 protein. Transfected cells were subjected to immunoprecipitation with an anti-HA antibody. The immunoprecipitated proteins and an aliquot of total lysate were electrophoresed and analyzed with anti-HA and anti-Myc antibodies. (B) mKRR was unable to upregulate the transcriptional activity of Nrf2. COS-1 cells were transfected with the expression vector for the indicated p21 protein, along with expression vectors for the NQO1-ARE firefly luciferase and TK-renilla luciferase. The reporter gene analysis was performed as described in Fig 2C. All error bars indicate standard deviations calculated from three independent experiments. *p<0.05 compared with wild-type. (C) Localization of endogenous Nrf2 and p21. Nrf2 and p21 in MDA-MB-231 cells were determined by double-label indirect immunofluorescence with anti-Nrf2 and anti-p21 antibodies. Colocalization of Nrf2 and p21 is indicated by the presence of yellow in the merge images. (D) Localization of p21 and its mutants. NIH 3T3 cells were cotransfected with HA-Nrf2 and each of the Myc-tagged p21 proteins. Double-label indirect immunofluorescence was performed with anti-HA and anti-Myc antibodies for detection of Nrf2 and p21, respectively.