The effects of HS on TNF-α–induced initiator caspase and Bcl2-interacting domain (Bid) activation. (A–G) MLE15 cells were incubated with 2 ng/ml TNF-α at 37°C or 42°C and sequentially lysed for fluorimetric (A, C, and E) and immunoblot (B, D, and F) analysis of caspase-8 (A and B), caspase-10 (C and D), and caspase-2 (E and F), and for immunoblot analysis of Bid (G). Caspase and Bid cleavage products are indicated by arrows (B, D, F, and G). Full-length Bid (Bid-FL) and caspases are indicated by large arrowhead. All graphs show means (±SE) of four experiments; *P < 0.05 versus 37°C. (B, D, F, and G) Each panel is representative of four similar blots. (H) MLE15 cells were pretreated with inhibitors of caspase-2, -8, and -10 (50 nM each) alone or in combination for 40 minutes at 37°C, then were incubated at 42°C with or without 2 ng/ml TNF-α and immunoblotted for activated caspase-3. A representative of three similar blots is shown. tBid, truncated Bcl2-interacting domain.