Membrane targeting is required for Bcl-B–induced apoptosis with Nur77. (A) HEK293T cells were transfected with 2 μg of either pEGFP-C1 or pEGFP-Nur77ΔDBD together with 0.5 to 1 μg plasmid DNA encoding a tagged Bcl-B or Bcl-BΔTM. After 24 hours, cells were lysed and caspase activity was assessed using DEVD-AFC substrate (mean ± SD; n = 3). (B) HEK293T cells were transfected as described for panel A. After 48 hours, fixed cells were stained with DAPI to visualize nuclei by UV microscopy. The percentages of apoptotic cells were determined by counting 200 GFP-positive cells (mean ± SE; n = 3). (C) HEK293T cells were transfected with pEGFP-C1 or pEGFP-Nur77ΔDBD in combination with Myc–Bcl-B or Myc–Bcl-BΔTM in the presence of 50 μM z-VAD-fmk. Cell lysates were prepared, and immunoprecipitation was performed with monoclonal anti-Myc monoclonal antibody. The immunoprecipitates or the lysates were immunoblotted with anti-GFP or anti-Myc antibodies.