Figure 4: Western blotting for examination of mitochondrial release of pro-apoptotic molecules and proteolytic activity in specific substrate cleavage. Treatments (7 days): CTL, ATRA (1.5 µg/kg/day), IFN-γ (5000 units/ kg/day), and ATRA (1.5 µg/kg/day) plus 4 hrs later IFN-γ (5000 units/kg/day). Representative Western blots show levels of β-actin, Smac/Diablo, AIF, calpain, caspase-9, and caspase-3. We examined the activities of calpain and caspase-3 in the cleavage of 270 kD α-spectrin to calpain-specific 145 kD spectrin breakdown product (SBDP) and caspase-3-specific 120 kD SBDP, respectively, in T98G xenograft. Combination therapy caused more increases in both 145 kD SBDP and 120 kD SBDP than control or monotherapy, indicating involvement of both calpain and caspase-3 activities in enhancing apoptosis. Results indicated involvement of caspase-dependent and caspaseindependent pathways in T98G xenograft for apoptosis after the combination therapy.