FIGURE 2.
BH3-M6, but not TPC, inhibits the binding of anti-apoptotic proteins to pro-apoptotic proteins in vitro. A, BH3-M6 blocks the binding of Bak BH3 α-helix to Bcl-XL (left) or Bim BH3 α-helix to Mcl-1 (right) as measured by fluorescence polarization assay, black circles: BH3-M6, open triangles: TPC. B, BH3-M6 inhibits the interaction of Bcl-XL or Mcl-1 with Bax and Bim as measured by GST pull-down assay. Purified GST, GST-Bcl-XL, and GST-Mcl-1 fusion proteins immobilized on glutathione-Sepharose beads were incubated for 1 h with 0, 50, and 100 μm BH3-M6 or 100 μm TPC, followed by addition of cell extracts from HEK293T or A549 cells for 1 h. Proteins associated with beads were eluted and analyzed by Western blotting with Bax and Bim antibodies. We used GST antibodies either for negative controls (in lysates incubated with GST beads) or to demonstrate equal input (in lysates incubated with GST-Bcl-XL or GST-Mcl-1 beads).