PMLRARα Interacts with Fas in acute promyelocytic leukemia (APL) cell line and APL primary cells. (A) Cell extracts of APL NB4 cells were immunoprecipitated using antibodies against green fluorescent protein (GFP) and Fas, or normal IgG and RARα followed by Western blot analysis with antibodies against Fas and RARα for detection of Fas and PMLRARα, respectively. The expression levels of Fas and PMLRARα in whole-cell extracts are also shown by Western blot. (B) Cell extracts of HL60 cells and HEK293 cells were immunoprecipitated using antibodies against GFP and Fas, IgG and PML, or Fas and IgG. The precipitates were subjected to immunoblot analysis using anti-Fas, anti-PML, and anti-RARα antibodies. The expression levels of Fas, PML and RARα in whole-cell extracts are also shown. (C) APL primary cells isolated from 2 patients were lysed and cell extracts were immunoprecipitated using antibodies against normal IgG and Fas. The precipitates were subjected to Western blot analysis using an anti-Fas antibody for detection of Fas, and anti-RARα antibody for detection of PMLRARα. The expression levels of Fas and PMLRARα in whole-cell extracts are shown on the bottom panels. (D) To assess location of Fas-PMLRARα complexes, APL NB4 cells were subjected to subcellular fractionation and the presence of proteins and complexes in the fractions was assessed by IP/WB analysis. Histone H3 and α-tubulin were used as nuclear and cytosol/membrane fraction control proteins, respectively.