BIM and PUMA are sequestered by BCL2, whereas newly synthesized BCL-X_L and BCL2A1 are not bound by BH3-only proteins. (A) The binding pattern of antiapoptotic BCL2 proteins to BH3-only proteins was investigated upon 0 to 16 hours of culture of CLL cells on CD154-expressing cells. BCL-X_L, BCL2A1, and NOXA are up-regulated at 4 to 16 hours, while levels of BCL2, MCL1, BIM, and PUMA are not altered (Lysate). IP of BCL2, BCL-X_L, MCL1, and BCL2A1 was done in lysis buffer containing 0.5% Triton X-100; however, to exclude the influence of detergent, IPs were repeated in CHAPS-containing lysis buffer with similar results (data not shown). Upon culture on CD154-expressing cells BIM and PUMAα were bound to BCL2 for up to 16 hours of culture, and up-regulated NOXA was bound by MCL1. In contrast, no binding of BH3-only proteins to BCL-X_L or BCL2A1 was detected (*nonspecific band). (B,C) Freshly isolated CLL cells were treated with 10 nM ABT-737 for 2 hours. After treatment, cells were lysed in CHAPS-containing lysis buffer. (B) IP of BCL2 revealed that upon treatment with ABT-737 the majority of BIM remained bound to BCL2 and only little BIM appeared in the supernatant (SN) of the IP. An interaction of BCL2 and BAK was found in 5 of 8 patients examined. Upon treatment with ABT-737, BAK was displaced from BCL2. (C) The reverse IP of BAK confirmed displacement of BAK from BCL2 by ABT-737.