PMLRARα recruits c-FLIP to Fas and inhibits procaspase-8 binding and cleavage. (A) U937/PR9 cells treated with or without 200μM ZnSO4 were activated with 50 ng/mL of CH-11 antibody and immunoprecipitated with anti-Fas antibody. The precipitants were analyzed for the presence of DISC subunits by Western blot (left panel). The panel on the right shows input protein levels in whole cell extracts. (B) APL NB4 cell extracts were immunoprecipitated using antibody against c-FLIP and control IgG antibody, and precipitates were Western blotted using anti-RARα and anti–c-FLIP antibodies (left panel). Equal input protein levels were confirmed by immunoblot analysis of β-actin in the whole cell lysates. APL primary cells from 2 patients were lysed and immunoprecipitated with anti-Fas or control IgG antibody (right panel). (This is the same experiment as shown in Figure 1C with extended analysis of c-FLIP). Precipitates were analyzed for the presence of PMLRARα and c-FLIP by Western blot. The quality of input material is shown in bottom panels. (C) Extracts from APL primary cells and HEK293 cells were subjected to Western blot analysis using anti–c-FLIP and anti–α-tubulin antibodies to compare relative protein expression levels. Quantification of bands with reference to tubulin levels was performed using Adobe Photoshop CS3 software. (D) Vehicle and arsenic trioxide-treated transgenic mouse APL cells (top) or scrambled shRNA cells and NB4 clone 4 cells (bottom) were treated with 0 or 50 ng/mL of FasL for 15 minutes. Whole cell lysates were then subjected to Western blot analysis using anti–caspase-8 and anti–β-actin antibodies. (E) NB4 clone 4 and scrambled shRNA NB4 cells were incubated with or without 10μM edelfosine, stained by propidium iodide and analyzed for the degree of apoptosis by flow cytometry.
