(A) Recombinant PB1-F2 protein caused release of cytochrome c from purified mouse mitochondria. Mouse mitochondria (50 μg total protein) were incubated for 1 h at 30 °C with recombinant protein of interest at indicated concentrations. The amount of cytochrome c released in the supernatant was assayed by immunoblotting using anti-cytochrome c antibody. Recombinant tBid protein was used as a control. The mitochondrial pellet was assayed for the Tom20 protein to ensure an equal amount of mitochondria was used.
(B) PB1-F2 and tBid cause dissipation of the mitochondrial membrane potential. Mouse mitochondria (50 μg) were incubated for 10 min at 30 °C with the protein of interest at indicated concentrations. Relative loss in membrane potential was measured by uptake of membrane potential-sensitive JC-1 dye. Data is presented as percent fluorescence relative to mock-treated mitochondria.
(C) PB1-F2 protein enhances tBid-induced cytochrome c release. Purified mouse mitochondria (50 μg) were incubated with 10 nM PB1-F2 or mock-treated for 15 min in 20 μl and were subsequently treated with recombinant tBid in the indicated concentrations for 1 h in a total volume of 30 μl. The supernatants were assayed for cytochrome c release by Western blot. The mitochondrial pellet was processed for Tom20 to ensure equal amount of mitochondria used.