PMLRARα-Fas binding domains and protection of mouse against Fas-induced death. (A) Schematic representation of PML deletion mutants. (B) HEK293 cells were transfected with pcDNA3-Flag vector, pcDNA3-Flag-PML-FL, or pcDNA3-Flag-deletion mutants illustrated in panel A. Cells lysates were immunoprecipitated using anti-Flag antibody (left) or anti-Fas antibody (middle). HEK293 cells were transfected with pcDNA3-Flag vector, pcDNA3-Flag-PML-FL or pcDNA3-Flag-PML-Del (B-box) mutant, lysed and immunoprecipitated with anti-Fas antibody (right). The precipitates were Western blotted with anti-Flag and anti-Fas antibody. (C) Schematic representation of Fas deletion mutants used to map binding to PMLRARα. Deletion of pre-ligand binding assembly domain (aa. 1-67) prevents trimerization of Fas deletion mutants with endogenous Fas. TM indicates transmembrane domain; and DD, death domain. (D) HEK293 cells were cotransfected with pcDNA3-PMLRARα, and plasmids expressing indicated YFP-tagged Fas mutants. Whole-cell lysates were immunoprecipitated using an anti-GFP antibody and the precipitates were Western blotted using anti-RARα and anti-GFP antibodies. The equal expression levels of PMLRARα in whole-cell extracts were confirmed by immunoblot analysis. (E) C57BL/6 mice were transfected with 100 μg of PMLRARα or empty vector plasmid using the hydrodynamic methodology.²²  Twenty-four hours later, the mice were inoculated intraperitoneally with a lethal dose of anti–mouse Fas antibody (Jo2) and monitored for survival 6 hours after challenge when the surviving mice were killed to harvest liver tissues for comparative analysis. (F) Representative images of the livers harvested at the time of death are shown. (G) Liver extracts of PMLRARα and vector-transfected mice were analyzed for PMLRARα-Fas complexes by immunoprecipitation and Western blotting using anti-RGSHis, anti-Fas, anti–c-FLIP, and anti–caspase-8 antibodies. (H) To confirm that PMLRARα blocks Fas-mediated apoptosis, the livers from panel E were subjected to immunohistochemical and TUNEL staining. Representative H&E-stained slides (top row), anti-RGS His antibody staining showing PMLRARα-RGSHis expression (2nd and 3rd rows), anti–cleaved caspase-3 antibody and TUNEL staining (4th and bottom row, respectively) are shown. (I) Model of PMLRARα-mediated inhibition of Fas apoptosis. PMLRARα in complex with Fas recruits FADD and c-FLIP and excludes procaspase-8 to block Fas-mediated apoptosis. At the same time, Fas complexed with PMLRARα is unable to bind FasL and CH-11. ASK1 indicates apoptosis signal-regulating kinase; FADD, Fas-associated death domain protein; JNK, c-Jun NH₂-terminal kinase; tBID, truncated Bid; CytC, cytochrome C; MKK7: mitogen-activated protein kinase kinase 7; and c-FLIP, cellular-FLICE inhibitory protein.