Figure 3.

PKCζ mediates ONOO⁻-induced p53 nuclear export and p53–Bcl-2 binding instead of the regulation of p53 transcriptional activity. (A and B) HUVECs were transfected with the p53-Luc reporter and Renilla luciferase–encoding plasmid (pRL- thymidine kinase) used as an internal control reporter together with p53–wild type or vector alone (pcDNA3.1; A). Some cells were further transfected with or without pcDNA3.1-CATζ (B). Transcriptional activity was determined by a reporter plasmid encoding 13 copies of the p53-binding sequence (p53-Luc reporter; Kern et al., 1992). After 24 h of transfection, p53 transcriptional activity was assayed using the dual-luciferase kit (B), or the cells were further treated with 10 or 50 µM ONOO⁻ for 8 h as indicated, and luciferase activity was assayed (A). Data are representative of triplicates using two or more different preparations of ECs. *, P < 0.05; **, P < 0.01. (C) HUVECs were transduced with Ad-DN-PKCζ or Ad-LacZ as a control for 24 h, treated with vehicle or 100 µM ONOO⁻ for 4 h, and immunostained with anti-p53 followed by DAPI counter staining for nuclei. Bar, 5 µm. (D, top) HUVECs were transduced with Ad-DN-PKCζ or Ad-LacZ for 24 h and stimulated with 100 µM ONOO^– for the indicated times. p53–Bcl-2 binding was determined by coimmunoprecipitation with anti-p53 followed by immunoblotting with anti–Bcl-2. p53, PKCζ, and Bcl-2 in total cell lysates were detected by Western blotting with each specific antibody. (bottom) Quantification of p53–Bcl-2 binding expressed as the relative band intensity ratio between coimmunoprecipitated versus total Bcl-2. Results were normalized as described in Fig. 1. n = 3. *, P < 0.05 and **, P < 0.01 compared with the vehicle control, and ^#, P < 0.05 and ^##, P < 0.01 compared with the LacZ control at each time point. Molecular masses are given in kilodaltons. Error bars indicate means ± SD. IB, immunoblot. IP, immunoprecipitation.