Sensitization of vav-Bcl-2 lymphocytes to ABT-737 requires the binding of Bim to Mcl-1. (A) Western blot analysis of NHL cell lines having higher or lower levels of Bcl-2 RNA by microarray analysis (Figure 1). Those expressing higher levels of Bcl-2 protein are sensitive to ABT-263 and express higher levels of Bim. (B) Expression of Bcl-2 and Bim in vavP–Bcl-2/Bim^+/+, vavP–Bcl-2/Bim^B/B, vavP–Bcl-2/Bim^N/N, and vavP–Bcl-2/Bim^P/P thymocytes was determined by Western blotting. (C) Thymocyte lysates were prepared and the protein complexes were analyzed by immunoprecipitation and Western blotting. (D) Affinities of Bim and Noxa BH3 peptides for mouse Bcl-2 and mouse Mcl-1 were determined by solution competition assay using a Biacore optical biosensor. Numbers in brackets represent standard deviations for n = 2 to 4 experiments. (E) The sensitivity of BM-derived B cells and thymocytes of the indicated genotypes to ABT-737 was measured and data are represented in a heat map as described in Figure 2. Results represent the mean of at least 3 independent experiments per genotype (detailed in supplemental Table 1). Restricting the binding specificity of Bim to that of Bad renders all cell types resistant to ABT-737, regardless of Bcl-2 overexpression. As a control, the BH3 mutations in Bim did not affect the resistance of vavP-Mcl-1–transgenic cells to ABT-737–induced cell death (vavP–Mcl-1/Bim^B/B and vavP–Mcl-1/Bim^N/N). (F) Thymocytes from vavP–Bcl-2/Bim^+/+ mice were treated with ABT-737 (1μM) for 5 hours before Bim immunoprecipitation. The composition of the protein complexes was analyzed by Western blotting.