Death receptor mediated cleavage of Bid in HeLa cells and cultured rat hippocampal neurons. Cleavage of FL-Bid in HeLa D98 cells (A)and cultured rat hippocampal neurons (B)exposed to TNF-α plus CHX (100 ng/ml plus 1 μg/ml) or staurosporine (STS, 3 μM and 300 nM, respectively) for the indicated time periods. Bid cleavage was detected by Western blot analysis. Duplicate experiments yielded similar results. (C)No detectable cleavage of FL-Bid during NMDA-induced neuronal death. Cultured rat hippocampal neurons were exposed to 300 μM NMDA in Mg2+-free HBS for 5 min, washed, returned to the original culture medium, and analyzed by Western blotting after 4 and 8 h. Sham-washed cultures were exposed to Mg2+-free HBS devoid of NMDA. Lack of Bid cleavage during excitotoxic neuron death was observed in four separate experiments. (D)Immunoblot analysis demonstrates significant cleavage of α-spectrin into its calpain-generated 150 and 145 kDa breakdown products. Duplicate experiments yielded similar results.