Figure 1.

NQO1 activity-dependent stabilization of p53 protein. (A) p53 null HCT116 cells were transfected with 150 ng of pRc/CMV human wild-type p53 with either pSG5 empty vector, 2 μg of pSG5 wild-type HA NQO1, or 3 μg of polymorphic HA C609T NQO1. (B) HCT116 (−) and HCT116 stably expressing HA NQO1 (+) cells were cultured without any treatments (N. T.), γ-irradiated (γ-IR) at 6 Gy, or treated with 100 μM H₂O₂. Cell extracts were prepared from untreated cells and from cells cultured for a half-hour and 4 h post-γ-irradiation and 6 h after the addition of H₂O₂. Protein extraction and immunoblot analysis were carried out as described in Materials and Methods by using PAb 1801 monoclonal anti-p53 antibody. The blots then were stripped and reprobed with monoclonal anti-Ha for the detection of HA NQO1 and with anti-actin antibody as a control for equal protein loading in each lane.