Figure 4.
Figure 4. PKCζ mediates d-flow–induced p53 SUMOylation and p53–Bcl-2 binding. (A) HeLa cells were transfected for 24 h as indicated with Flag-tagged p53, HA-tagged SUMO3, and HA-tagged CATζ. p53 SUMOylation was detected by immunoprecipitation with anti-Flag followed by Western blotting with anti-SUMO2/3 (top). Both protein expression and immunoprecipitated p53 were confirmed by anti-Flag antibody, and CATζ and SUMO expression were detected with anti-HA. Mono-SUMOylation band (∼74 kD) and poly-SUMOylation bands (>78 kD) were detected. The asterisk indicates mono-SUMOylation of p52. (B) HUVECs were transfected for 24 h with either PIASy or control siRNA as indicated, and then the cells were transfected with HA-CATζ or vector alone for another 24 h. (top) p53 SUMOylation was detected by immunoprecipitation with anti-p53 followed by Western blotting with anti-SUMO2/3. PIASy expression was confirmed by immunoblotting with anti-PIASy, and p53, HA-CATζ, and SUMO expression was confirmed with anti-p53, -HA, and -SUMO2/3, respectively. (C) HUVECs were transfected with either p53 or control siRNA as indicated for 24 h, and then the cells were transduced with an Ad-SENP2 or LacZ with a control for another 24 h. p53 SUMOylation, expression of p53, SENP2, and SUMO were determined as described in Materials and methods. The asterisks indicate nonspecific bands. (D) HUVECs were transduced with Ad-DN-PKCζ or Ad-LacZ as a control for 24 h and then stimulated with d-flow for the indicated times. p53 SUMOylation and p53–Bcl-2 binding were determined as described in Materials and methods. (left graph) Intensities of SUMOylated p53 bands at 74, 82, 130, and 185 kD were quantified by densitometry after subtracting background gel density. After normalization of each control as described in Fig. 1, results were expressed relative to the SUMOylation level in static condition (0 min; 100%). Shown are means ± SD (n = 3). **, P < 0.01 compared with the vehicle control or the LacZ control at each time point. (E) HUVECs were transfected with either PKCζ or control siRNA as indicated for 24 h and then stimulated with d-flow for 3 h. p53 SUMOylation, p53–Bcl-2 binding, expression of p53, Bcl-2, SUMO, and various PKC isoforms as indicated were determined as described in Materials and methods. Immunoblots are representative of three separate experiments. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation. KR, K386R. WT, wild type.

PKCζ mediates d-flow–induced p53 SUMOylation and p53–Bcl-2 binding. (A) HeLa cells were transfected for 24 h as indicated with Flag-tagged p53, HA-tagged SUMO3, and HA-tagged CATζ. p53 SUMOylation was detected by immunoprecipitation with anti-Flag followed by Western blotting with anti-SUMO2/3 (top). Both protein expression and immunoprecipitated p53 were confirmed by anti-Flag antibody, and CATζ and SUMO expression were detected with anti-HA. Mono-SUMOylation band (∼74 kD) and poly-SUMOylation bands (>78 kD) were detected. The asterisk indicates mono-SUMOylation of p52. (B) HUVECs were transfected for 24 h with either PIASy or control siRNA as indicated, and then the cells were transfected with HA-CATζ or vector alone for another 24 h. (top) p53 SUMOylation was detected by immunoprecipitation with anti-p53 followed by Western blotting with anti-SUMO2/3. PIASy expression was confirmed by immunoblotting with anti-PIASy, and p53, HA-CATζ, and SUMO expression was confirmed with anti-p53, -HA, and -SUMO2/3, respectively. (C) HUVECs were transfected with either p53 or control siRNA as indicated for 24 h, and then the cells were transduced with an Ad-SENP2 or LacZ with a control for another 24 h. p53 SUMOylation, expression of p53, SENP2, and SUMO were determined as described in Materials and methods. The asterisks indicate nonspecific bands. (D) HUVECs were transduced with Ad-DN-PKCζ or Ad-LacZ as a control for 24 h and then stimulated with d-flow for the indicated times. p53 SUMOylation and p53–Bcl-2 binding were determined as described in Materials and methods. (left graph) Intensities of SUMOylated p53 bands at 74, 82, 130, and 185 kD were quantified by densitometry after subtracting background gel density. After normalization of each control as described in Fig. 1, results were expressed relative to the SUMOylation level in static condition (0 min; 100%). Shown are means ± SD (n = 3). **, P < 0.01 compared with the vehicle control or the LacZ control at each time point. (E) HUVECs were transfected with either PKCζ or control siRNA as indicated for 24 h and then stimulated with d-flow for 3 h. p53 SUMOylation, p53–Bcl-2 binding, expression of p53, Bcl-2, SUMO, and various PKC isoforms as indicated were determined as described in Materials and methods. Immunoblots are representative of three separate experiments. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation. KR, K386R. WT, wild type.

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