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Figure S1. Description of BH3 peptides and their binding affinities for four prosurvival proteins. (A) Sequence comparison of wild-type and mutated BH3 peptides used in this study. Each is from the human sequence unless indicated as being from the mouse (e.g., mBmf). Amino acid numbering is indicated in parentheses, and mutated residues are indicated in bold italic. Boxes indicate the highly conserved leucine and aspartate residues. The peptides used in this study were synthesized by Mimotopes (Victoria, Australia), purified by reverse-phase HPLC, and dissolved as 1-2-mM stock solutions in water. The GenBank/EMBL/DDBJ accession nos. for the peptides were as follows: human Bim_L (AAC39594), mouse Bim_L (AAC40030), human Puma (AAK39542), mouse Bmf (AAK38747), human Bad (NP_004313), human Bik (NP_001188), human Hrk (NP_003797), human Bid (NP_001187), human Noxa (NP_066950), and mouse Noxa (NP_067426). Mutant Bim or Noxa peptides have been previously described (Wilson-Annan, J., L.A. O’Reilly, S.A. Crawford, G. Hausmann, J.G. Beaumont, L.P. Parma, L. Chen, M. Lackmann, T. Lithgow, M.G. Hinds, et al. 2003. J. Cell Biol. 162:877-888; Chen, L., S.N. Willis, A. Wei, B.J. Smith, J.I. Fletcher, M.G. Hinds, P.M. Colman, C.L. Day, J.M. Adams, and D.C.S. Huang. 2005. Mol. Cell. 17:393-403). (B) Relative binding affinity (IC50; nanomolars) of BH3 peptides for prosurvival proteins. Analysis of protein interactions was performed on a biosensor (model 3000; Biacore) as previously described (Hinds, M.G., M. Lackmann, G.L. Skea, P.J. Harrison, D.C.S. Huang, and C.L. Day. 2003. EMBO J. 22:1497-1507; Wilson-Annan, J., L.A. O’Reilly, S.A. Crawford, G. Hausmann, J.G. Beaumont, L.P. Parma, L. Chen, M. Lackmann, T. Lithgow, M.G. Hinds, et al. 2003. J. Cell Biol. 162:877-888; Chen, L., S.N. Willis, A. Wei, B.J. Smith, J.I. Fletcher, M.G. Hinds, P.M. Colman, C.L. Day, J.M. Adams, and D.C.S. Huang. 2005. Mol. Cell. 17:393-403). Most of the data for Bcl-x_L, Bcl-w, and Mcl-1 have been reported previously (Chen, L., S.N. Willis, A. Wei, B.J. Smith, J.I. Fletcher, M.G. Hinds, P.M. Colman, C.L. Day, J.M. Adams, and D.C.S. Huang. 2005. Mol. Cell. 17:393-403). The binding affinity of prosurvival proteins with a shorter C-terminal truncation (e.g., Bcl-wΔC10), as used in the present mitochondrial studies, were typically found to be around twofold weaker for each peptide tested (unpublished data), which is consistent with previous findings that the removal of C-terminal residues facilitates the binding of BH3-only proteins to the hydrophobic groove but decreases attachment to mitochondria (Hinds, M.G., M. Lackmann, G.L. Skea, P.J. Harrison, D.C.S. Huang, and C.L. Day. 2003. EMBO J. 22:1497-1507; Wilson-Annan, J., L.A. O’Reilly, S.A. Crawford, G. Hausmann, J.G. Beaumont, L.P. Parma, L. Chen, M. Lackmann, T. Lithgow, M.G. Hinds, et al. 2003. J. Cell Biol. 162:877-888; Day, C.L., L. Chen, S.J. Richardson, P.J. Harrison, D.C.S. Huang, and M.G. Hinds. 2005. J. Biol. Chem. 280:4738-4744). Mouse and human BadBH3 have similar binding profiles (unpublished data). Certain data relating to specific interactions between prosurvival and BH3-only proteins is also in schematic form in Fig. 1 B.