S100 fractions from untreated cells (MDA‐MB‐468) or from cells treated with direct Akt inhibitors (MK‐2206 [1 μM], A‐443654 [0.4 μM]) or Akt pathway inhibitors (LY294002 [25 μM], deguelin [10 nM]) were isolated. Akt was immunodepleted by sequential immunoprecipitation in untreated S100 fractions, and the efficiency of immunodepletion was tested by immunoblot analysis. IgG was used as a negative control for immunodepletion experiments. The indicated S100 fractions were incubated with MDA‐MB‐435 mitochondrial preparations, and change in priming was assessed by using BH3 profiling. Responses to various BH3 peptides are shown. The phosphatase inhibitor cocktail PhosSTOP was used in all buffers. Cytochrome
c release was determined by ELISA. Bars indicate the mean of three independent experiments (
n = 3). Symbols indicate the mean of at least two technical replicates for each independent experiment. Two‐way ANOVA was conducted on the influence of two independent variables (BH3 peptide, treatment) on cytochrome
c release of isolated mitochondria from ABT‐737‐sensitive MDA‐MB‐435 cells that were treated with the S100 fraction isolated from ABT‐737‐resistant MDA‐MB‐468 or ZR‐75‐1 cells. Each treatment was statistically compared to control within each peptide group using
t‐tests with Bonferroni correction for multiple comparisons. See
Appendix Table S2 for
P‐values.