In vivo interaction profile of all 6 prosurvival Bcl-2 proteins with all BH3-only proteins, Bax, and Bak. (A-B) Human embryonic kidney 293T cells were cotransfected to express each HA-tagged human prosurvival Bcl-2 protein or empty vector (E.V.) control in combination with each of the indicated Myc-tagged human BH3-only proteins (A), Myc-tagged human Bak, or Flag-tagged constitutively active (L161P) human Bax (B) in the presence of Q-VD-OPH (10 µM). After 24 hours, cells were fixed with PFA to maintain protein-protein interactions, lysed with buffer containing 1% CHAPS, and subjected to immunoprecipitation with anti-HA mAb. Total cell lysates (TCL) and immunoprecipitates (IP) were analyzed by western blotting with anti-(α)HA, α-Myc, or α-Flag mAbs. Blots are representative of 3 independent experiments. Asterisks denote the light chain of the mAb used for immunoprecipitation. (C) Signals from western blots as presented in panel A-B were quantified and the relative affinity of the proapoptotic Bcl-2 proteins for each of the prosurvival Bcl-2 proteins was expressed in arbitrary units (AU), in a normalized manner, as described in supplemental “Materials and methods.” Data are derived from 3 independent experiments. (D) Schematic representation of binding affinities calculated in panel C. Red, <0.1; yellow, <0.5; green, >0.5.