Fig. 5. Restoration of TNF sensitivity in ASK1-re-introduced ASK1–/– MEFs. (A) Restoration of TNF-induced sustained activation of JNK. ASK1–/– MEFs were infected with adenoviruses encoding β-galactosidase (LacZ) or HA-tagged wild-type ASK1 (Saitoh et al. 1998) for 24 h. Cells were treated with TNF (50 ng/ml for 120 min), and cell lysates were subjected to westen blot analysis with phospho-JNK antibody. Essentially the same relsults were obtained for H₂O₂-treatment. Appropriate expression of exogenous ASK1 is shown by anti-HA blotting. (B) Restoration of TNF-induced apoptosis. ASK1–/– MEFs were infected as described above, treated with TNF (50 ng/ml in the presence of 1 µg/ml CHX) for 6 h, and the percentage of TUNEL-positive cells was calculated as described in Figure 4O. Values are means (± SD) of three random fields (at least 100 cells per field counted).