FIGURE 5.

Bax activation and subsequent apoptotic cell death provoked by Bcl-xL deficiency can proceed in absence of Bid and Bim. A, Western blot of platelet lysates for the expression of Puma, Bcl-2, Bcl-w, and Bcl-xL. WT and Bid−/−Bim−/− stand for wild type and bid^−/−bim^−/−, respectively. B–E, platelets (1.0 × 10⁸) isolated from Bid/Bim double knock-out mice were incubated with 10 μm ABT-737 or vehicle for 2 h at room temperature. B, Western blot for the expression of Bax after immunoprecipitation (IP) using mouse antibody that specifically recognizes activated Bax (6A7) or mouse control IgG. C, Western blot for the expression of Bak, Bax, CoxIV (cytochrome c oxidase IV), and GAPDH after cellular fractionation of the platelet lysates. D, Western blot for the expression of Bax after incubation of the platelet lysates with protein cross-linkers (5 mm bismaleimidohexane and 5 mm bis(sulfosuccinimidyl) suberate). E, Western blot of platelet lysates for the expression of cleaved caspase-3. F and G, platelets (2.0 × 10⁶) isolated from Bid/Bim double knock-out mice were incubated with 10 μm ABT-737 or vehicle for 2 h at room temperature. F, caspase-3/7 activity of platelet supernatant (n = 4/group). G, MTS assay (n = 5/group). IB, immunoblot; RLU, relative light units.