Fig. 2. Oxidative stress enhances the interaction between PP5 and ASK1. (A) H₂O₂- and TNF-induced interaction of PP5 and ASK1. HeLa cells were transiently co-transfected with HA-PP5 and Flag-ASK1. Thirty-six hours later, the cells were treated with 1 mM H₂O₂ or 200 ng/ml TNF for 20 min, and lysates were subjected to co-immunoprecipitation analysis as described in Figure 1A. (B) H₂O₂ dose-dependent interaction of PP5 and ASK1. HeLa cells and COS7 were transfected as in (A), treated with increasing concentrations of H₂O₂ for 30 min and analyzed by co-immunoprecipitation analysis. (C) Time course of the H₂O₂-induced interaction of PP5 and ASK1. HeLa cells were transfected as in (A), treated with indicated concentrations of H₂O₂ for the indicated periods and analyzed by co-immunoprecipitation analysis. (D) Specific interaction of ASK1 with PP5 but not PP2A. 293 cells were transiently co-transfected with ASK1-HA and Flag-PP5 or Flag-PP2A. Cells were treated with 5 mM H₂O₂ for 30 min, and lysates were subjected to co-immunoprecipitation analysis as described in Figure 1A. (E) Interaction of endogenous PP5 and ASK1. Approximately 5 × 10⁷ of A549 cells were treated with 5 mM H₂O₂ for 30 min. Cell lysates were divided and immunoprecipitated with normal rabbit IgG or anti-ASK1 polyclonal antibody (DAV) and were immunoblotted with anti-PP5 monoclonal antibody. The presence of ASK1 and PP5 in the same lysates was verified by immunoblotting (WB). Ig indicates non-specific reactions derived from rabbit IgG. (F) Subcellular localization of endogenous PP5 in A549 cells. A subcellular fractionation was performed as described in Materials and methods, and PP5 was detected by immunoblotting. Anti-PML antibody was used as a positive control for the nuclear protein. (G) Subcellular localization of transfected ASK1 and PP5 in HeLa cells. HeLa cells were transfected with Flag-ASK1 and HA-PP5, or with HA-PP5 alone, and the cells were subjected to an immunofluorescence staining as described in Materials and methods.