Analysis of extrinsic and intrinsic pathways after treatment with BB-1 and TRAIL. KOB cells (5 × 10⁵/mL) were incubated for 48 hours with 0.5 μg/mL TRAIL [T], 12.5 μg/mL BB-1 [B], BB-1+TRAIL (simultaneous addition [B+T]), or BB-1+TRAIL (0.5 μg/mL TRAIL was added after incubation with 12.5 μg/mL BB-1 for 24 hours [B→T]), or without reagents [C]. Cells were harvested at the indicated time points and analyzed. (A-B,D) Western blot analysis. Using 30 μg cell extract, caspases and proapoptotic and antiapoptotic factors were detected with the antibodies described in “Materials and methods.” (C) Mitochondrial membrane potential (ΔΨm) and cytochrome c translocation. After treatment, cells were evaluated using DiOC6 by FCM, and loss of ΔΨm (%) is indicated in each panel. Using 10 μg cytosolic extract, cytochrome c was detected by Western blotting with the antibodies described in “Materials and methods.”