Some Bcl-2 amino acid substitutions increase affinity for proapoptotic family members. (A) Binding between immobilized human Bim BH3 peptide and 300 nM WT Bcl-2 or the G33R variant was analyzed by surface plasmon resonance. (B) Summary of dissociation constants for complexes of WT or mutant Bcl-2 with Bim BH3 peptide, Puma BH3 peptide, or BakΔTM protein showing that Bim and Puma BH3 domains are bound more tightly to Bcl-2 G33R or A43T than to WT. Error bars, SD of 3 independent experiments using different chips and protein preparations. *P < .03 vs WT sequence. (C) After cDNA encoding WT or variant Bcl-2 fused to S-peptide was expressed for 24 hours in Jurkat cells, S-peptide pulldowns (top) or whole cell lysates (bottom) were probed for S-peptide or the indicated Bcl-2 binding partners. (D) Nalm6 or Jurkat cells were transfected with plasmid encoding enhanced green fluorescent protein (EGFP) or EGFP fused to Bim_EL,⁵⁹  along with empty vector or plasmid encoding the indicated Bcl-2 variant. Cells were cultured for 24 hours in the presence of the broad spectrum caspase inhibitor Q-VD-OPh,⁶⁰  and subjected to immunoblotting to assess expression (left panels). Alternatively, after culture for 24 hours in the absence of Q-VD-OPh for 24 hours, cells were stained with APC-conjugated Annexin V and subjected to 2-color flow cytometry. Right panels show summary of EGFP-positive/Annexin V-negative cells (as a percentage of total cells) after transfection of Nalm6 cells (upper panel) or Jurkat cells (lower panel). Error bars, ± SD of 3 independent experiments. Procedures used for these in vitro studies were previously published^46,47,53,61  and are described in the supplemental Methods.