Inhibition of apoptosis and enhancement of NF-κB/Rel nuclear levels in 32D cells by amifostine. (A) 32D cells (2 × 10⁵/mL) were incubated in the presence or the absence of WEHI-3 CM and with or without amifostine (100 μg/mL). Propidium iodide incorporation in cell DNA and caspase 3 activity were measured after 18 and 12 hours, respectively. (B) 32D cells (2 × 10⁵/mL) were incubated in the presence or absence of WEHI-3 CM and with or without amifostine (100 μg/mL) for 3 hours. The levels of IκB were analyzed in cytoplasmic extracts by Western blotting. The NF-κB/Rel complexes were examined by EMSA of nuclear extracts incubated with a ³²P-labeled κB oligonucleotide. Where indicated, the nuclear extracts were incubated with the³²P-labeled κB oligonucleotide in the presence of excess (50×) amounts of unlabeled κB or a different (NF-AT) oligonucleotide.