Dissociation of Bax from Bcl-X
by DJ-1(L166P). (A-B) Supernatant containing 50 μg of recombinant His-Bax (A) or Bcl2 (B) was incubated with 20 μg of GST or GST-Bcl-XL coupled to glutathione-agarose beads. After being washed to remove unbound proteins, the glutathione-agarose beads were incubated with various amounts of His-DJ-1(L166P) supernatant. The bound proteins were subjected to immunoblot analysis. (C) H1299 cells stably expressing EGFP-Bcl-XL or EGFP-Bcl-XL(1–195) were transiently transfected with various amounts of Flag-DJ-1(L166P) and treated with 80 mJ/cm2 UVB irradiation. The supernatants of cell lysates were subjected to immunoprecipitation with anti-GFP antibodies. (D) The relative levels of Bax bound to EGFP-Bcl-XL in (D) were analyzed using densitometry (mean ± S.E.M., n = 3; *, p <0.05; **, p <0.01; ***, p <0.001; by one-way ANOVA).