Nur77 binds selectively to Bcl-2, Bfl-1, and Bcl-B. (A) GST fusion proteins representing Bcl-2 members (2 μg) were incubated overnight at 4°C with lysates from HEK 293T cells transfected with plasmid encoding GFP-Nur77. GST proteins were recovered on glutathione-Sepharose, and associated proteins were analyzed by SDS-PAGE immunoblot using rabbit anti-GFP (top blot) and anti-GST (bottom blot) antibodies. (B) 293T cells were cotransfected with plasmids encoding either GFP or GFP-Nur77ΔDBD (a mutant of Nur77 lacking the DNA binding domain that allows Nur77 to enter the cytoplasm without requiring additional stimulation) in combination with plasmids encoding Myc-tagged versions of various Bcl-2 family members. After 24 hours, immunoprecipitation (IP) was performed using anti-Myc antibody, and immune-complexes were analyzed by immunobloting using anti-GFP (top blot) or anti-Myc (middle blot) antibodies. Cell lysates (50 μg) were analyzed directly (bottom blot). (C) For Bcl-W, we performed immunoprecipitation using anti–Bcl-W antibody, and immune-complexes were analyzed by immunobloting using anti-GFP (top blot) and anti–Bcl-W (middle blot) antibodies. BimEL serves as a positive control. Lysates (50 μg) were also analyzed directly in gels to confirm protein production (“total lysate”) (bottom). Asterisks indicate nonspecific bands.