Figure 3
High levels of active initiator caspase-9 in FDD KI mice. A, Homogenates (input) were prepared from the bVAD injected (+bVAD) and contralateral non-injected (con.) hippocampal regions of WT and FDDKI mice. Active caspases were isolated from homogenates with streptavidin-agarose-beads pull-down. Western blot analysis shows that the caspase inhibitor bVAD traps FL-caspase-9 only from the bVAD injected FDDKI mouse hippocampus; FL-caspase-8, cl.caspase-3 and cl.-caspase-6 are not trapped. B, In a similar experiment, the streptavidin-agarose-beads pull-down experiment was performed from the P2 fractions. The P2 fractions represent crude synaptosomal fractions (see Material and Methods for details about these preparations). Again, active FL-caspase-9 is isolated from FDDKI but not WT mice. C, Organotypic hippocampal cultures from either FDDKI or WT mice were incubated for 3 hrs with 45 μM bVAD. After lysis, active caspases were isolated from homogenates. Again, caspase-9 was the only active caspase isolated. Albeit traces of active caspase-9 are found in the WT samples, the levels found in the FDDKI hippocampus are greatly elevated. The blots shown in A, B and C are representative of duplicate experiments.