Fig. 3. Both the RING finger and the zinc finger domain are required for full activation of JNK and p38 in MEF cells. MEF cells were derived from wild-type (+/+) or TRAF6^–/– (–/–) mice. (A) Activation of JNK. Cells were either unstimulated (–) or stimulated (+) with IL-1 (20 ng/ml) for 15 min or LPS (500 ng/ml) for 90 min. After stimulation, cells were assayed for endogenous JNK activity by immunocomplex kinase assay with GST–c-Jun (1–89) as a substrate (GST–c-Jun). The amount of JNK in the immunocomplex is also shown (JNK). The fold increase in endogenous JNK activity in stimulated cells compared with untreated cells was determined by phosphoimaging and normalized to the level of JNK in the immunocomplex, which was determined by immunoblotting using ImageQuant (Molecular Dynamics). (B) Activation of p38 MAPK. After stimulation as described in (A), cells were lysed with sample buffer. p38 MAPK (p38) and phosphorylated p38 (p-p38) were visualized by immunoblotting with anti-p38 and anti-p-p38 antibodies. The fold increase in the amount of p-p38 in stimulated cells compared with unstimulated cells was determined and normalized to the amount of p38 as measured by ImageQuant.