The lysineless Bcl-B K/R mutant behaves like WT Bcl-B in terms of localization and interactions with BH3-only proteins. (a) Subcellular localization. U2OS cells transfected to express HA-Bcl-B and Life-Act-GFP were stained to detect nuclei (DAPI, 4',6-diamidino-2-phenylindole), mitochondria (Mitotracker Deep Red) and Bcl-B (anti-HA), and examined by confocal laser-scanning microscopy. Representative cells of multiple independent experiments are shown. (b) Colocalization of Mitotracker and HA-Bcl-B signal of experiments such as shown in a was quantified using the Intensity Correlation Analysis plugin and ImageJ software. A total of 80 or 96 cells were analyzed for Bcl-B WT and Bcl-B K/R, respectively. (c) Interaction with BH3-only proteins. HEK 293 T cells were transfected to express WT or K/R mutant HA-Bcl-B, together with Myc-tagged Bik, Bim, Puma or Noxa. Cells were lysed in CHAPS buffer and Bcl-B was isolated by IP with anti-HA mAb. Total cell lysates (TCL) and precipitates (IP) were analyzed by immunoblotting with anti-α-HA or α-Myc antibody. Data shown are representative of multiple independent experiments.