Titrating Mcl-1 exogenous protein to find out the condition in which both Bad and Noxa BH3 peptides exhibit their binding selectivity. 2LMP mitochondria were pre-treated with buffer alone or recombinant anti-apoptotic Bcl-2 proteins at low concentration (A), at escalated concentration (B, C) before being permeablized by Bim BH3 peptide together with Bad or Noxa BH3 peptides at indicated concentrations. 2LMP mitochondria pre-treated with buffer alone or 200 nM of Bcl-2, Bcl-xL or Mcl-1 were incubated with different concentrations of Bim BH3 peptide which antagonized all three anti-apoptotic proteins to recover Cytochrome C and Smac release induced by 25 nM of Bim BH3 peptide (D). Mitochondrial pellets (P) and supernatants (S) were carefully separated and mixed with sample buffer, and equivalent portions were subjected to SDS-PAGE and Western blotting analysis for Cytochrome C and Smac/Diablo. Results are representative of three independent experiments. N*: Noxa BH3 peptide.