Figure 8. PKCζ–PIASy association is critical for p53 SUMOylation and p53–Bcl-2 binding. (A) HUVECs were stimulated with 100 µM ONOO– for the indicated times and subjected to immunoprecipitation with anti-PIASy followed by Western blotting with anti-PKCζ (top). (B and C) Association between PKCζ and PIASy was tested by a mammalian two-hybrid assay. HeLa cells were transfected with plasmids containing Gal4-PKCζ wild type and VP16-PIASy (B) or truncated mutants of VP16-PIASy (C) as well as the Gal4-responsive luciferase reporter pG5-luc. After 24 h of transfection, cells were stimulated with 100 µM ONOO− or vehicle for 16 h, and luciferase activity was quantified. Luciferase activity was normalized with the Renilla luciferase (Luc.) activity (Woo et al., 2008). Data are representative of three experiments using two or more different preparations of ECs (means ± SD; **, P < 0.01). (D) PIASy binding to PKCζ occurs via a domain consisting of aa 301–410 of PIASy. HeLa cells were transfected with each of the Flag-tagged PIASy fragments, and then pull-down assays were preformed using anti-Flag and IgG Sepharose beads in the presence of GST-fused recombinant PKCζ. Association of PIASy fragments with GST-PKCζ was assayed by Western blotting with anti-PKCζ. (bottom) PIASy fragment expression was detected by Western blotting with anti-Flag. (E) PIASy Fr3, but not Fr4, inhibited PKCζ–PIASy association. HUVECs were cotransfected with HA-tagged PKCζ wild type, Myc-tagged PIASy wild type, and Flag-tagged PIASy Fr3 or Fr4 for 24 h. Myc-PIASy wild type was immunoprecipitated with anti-Myc followed by immunoblotting with anti-HA (top). The expression of PKCζ, PIASy, and PIASy fragments was detected by Western blotting with specific antibodies. Data are representative of three independent experiments. (F) HUVECs were transfected with Flag-tagged PIASy Fr3 or Fr4 or control vectors for 24 h and then stimulated by d-flow for 3 h. p53 was immunoprecipitated using anti-p53, and d-flow–induced p53 SUMOylation was analyzed by immunoblotting with anti-SUMO2/3 (top). The expression of p53, SUMO, and PIASy fragments was detected by Western blotting with specific antibodies. Data are representative of three independent experiments. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation. WT, wild type.
Figure 8.

PKCζ–PIASy association is critical for p53 SUMOylation and p53–Bcl-2 binding. (A) HUVECs were stimulated with 100 µM ONOO– for the indicated times and subjected to immunoprecipitation with anti-PIASy followed by Western blotting with anti-PKCζ (top). (B and C) Association between PKCζ and PIASy was tested by a mammalian two-hybrid assay. HeLa cells were transfected with plasmids containing Gal4-PKCζ wild type and VP16-PIASy (B) or truncated mutants of VP16-PIASy (C) as well as the Gal4-responsive luciferase reporter pG5-luc. After 24 h of transfection, cells were stimulated with 100 µM ONOO− or vehicle for 16 h, and luciferase activity was quantified. Luciferase activity was normalized with the Renilla luciferase (Luc.) activity (Woo et al., 2008). Data are representative of three experiments using two or more different preparations of ECs (means ± SD; **, P < 0.01). (D) PIASy binding to PKCζ occurs via a domain consisting of aa 301–410 of PIASy. HeLa cells were transfected with each of the Flag-tagged PIASy fragments, and then pull-down assays were preformed using anti-Flag and IgG Sepharose beads in the presence of GST-fused recombinant PKCζ. Association of PIASy fragments with GST-PKCζ was assayed by Western blotting with anti-PKCζ. (bottom) PIASy fragment expression was detected by Western blotting with anti-Flag. (E) PIASy Fr3, but not Fr4, inhibited PKCζ–PIASy association. HUVECs were cotransfected with HA-tagged PKCζ wild type, Myc-tagged PIASy wild type, and Flag-tagged PIASy Fr3 or Fr4 for 24 h. Myc-PIASy wild type was immunoprecipitated with anti-Myc followed by immunoblotting with anti-HA (top). The expression of PKCζ, PIASy, and PIASy fragments was detected by Western blotting with specific antibodies. Data are representative of three independent experiments. (F) HUVECs were transfected with Flag-tagged PIASy Fr3 or Fr4 or control vectors for 24 h and then stimulated by d-flow for 3 h. p53 was immunoprecipitated using anti-p53, and d-flow–induced p53 SUMOylation was analyzed by immunoblotting with anti-SUMO2/3 (top). The expression of p53, SUMO, and PIASy fragments was detected by Western blotting with specific antibodies. Data are representative of three independent experiments. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation. WT, wild type.

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