Figure 2.
BKA-073 specifically binds to Bak, induces Bak oligomerization and Cyt c release in lung cancer cells. (A) Fluorescence polarization assay was performed to measure the inhibitory constant (Ki) value using purified Bak protein or other Bcl2 family member(s), BKA-073, and fluorescence-labeled Bak BH3 peptide. Data represent the mean ± SD, n = 3 per group. (B) The binding affinity of BKA-073 with WT Bak or ∆BH3 Bak deletion mutant protein was examined by isothermal titration calorimetry assay. The binding constant (KD) value was determined by fitting of the titration curve to a 1-site binding mode. (C) A549 cells were treated with BKA-073 (1μM) for 24h, followed by mitochondrial isolation and crosslinking using BMH. Bak was analyzed by western blot. (D) A549 cells were treated with increasing concentrations of BKA-073 for 24h. Mitochondrial and cytosolic fractions were isolated. Levels of Cyt c in these two fractions were analyzed by Western blot. Prohibitin was used as a mitochondrial marker for purity of the subcellular fractions.