Requirement of the C-terminus of Bcl-X
for DJ-1(L166P) binding. (A) The supernatant of E. coli crude extract containing recombinant His-DJ-1 or His-DJ-1(L166P) was incubated with GST, GST-Bcl-XL, GST-Bcl-XL(1–85), GST-Bcl-XL(86–195) or GST-Bcl-XL(196–233) coupled to glutathione-agarose beads. The bound proteins were subjected to immunoblot analysis. (B) The supernatants of H1299 cells transfected with EGFP, EGFP-Bcl-XL, EGFP-Bcl-XL(1–195) or EGFP-Bcl-XL(196–233) along with Flag-DJ-1(L166P) were subjected to immunoprecipitation with anti-GFP antibodies. (C) H1299 cells transfected with si-NC or si-DJ-1 along with Flag, Flag-DJ-1(s) or Flag-DJ-1(L166P)(s) were treated with or without 80 mJ/cm2 UVB irradiation. Total cell lysates were subjected to immunoblot analysis. (D) The relative ratios of Bcl-XL to α-Tubulin in (C) were analyzed using densitometry (mean ± S.E.M., n = 3; **, p <0.01; ns, no statistical significance by one-way ANOVA). (E) H1299 cells transfected with EGFP or EGFP-Bcl-XL along with Flag, Flag-DJ-1 or Flag-DJ-1(L166P) were treated with 80 mJ/cm2 UVB and then with 5 μM Lactacystin for 16 hours. The supernatants of the cell lysates were subjected to immunoprecipitation with anti-GFP antibodies.