Fig. 9. Impaired apoptotic response of mutant keratinocytes exposed to TNF. Primary keratinocytes from newborn mutants (Mut) and wild-type (WT) littermates were exposed to various concentrations of TNF in the presence of cycloheximide (CHX), an inhibitor of protein translation (Caulin et al., 2000). (a) Western blot detection of pro-caspase 3 and active caspase 3. ß-Actin was used as a loading control. (b) Quantitative evaluation of the western blot signals shown in panel a. The relative amount of active caspase 3 (normalized against ß-actin) in the two genotypes is shown as a function of the TNF concentration. Note the reduced apoptotic response of mutant cells compared with wild-type controls at a TNF concentration 10 ng/ml. This experiment was repeated twice with independent isolates of newborn keratinocytes from mutant and wild-type littermates. Similar results were obtained in these experiments (data not shown) (P=0.04).