Panel A - RAW cells were pre-treated for 60 min without (-) or with (+) 10 μM U0126 (U0) or 2 μM PD 184352 (PD) and then either left untreated or stimulated for 45 min with 100 ng/ml LPS. Cells were then lysed and subjected to SDS-Page analysis before being transferred to nitrocellulose membrane and blotted with the appropriate antibody. The upper panels were probed with anti-TACE phospho Thr 735 at 1 μg/ml in the presence of 10 μg/ml non-phosphorylated peptide, while the lower panels were probed with an antibody that recognises the phosphorylated and unphosphorylated forms of TACE equally well [anti-mouse TACE, Spring Biosciences]. Panel B – BMDM from wildtype (WT) of Tpl-2-deficient mice (Tpl-2 -/-)were either left untreated or stimulated with 100 ng/ml LPS for the times indicated. Immunoblotting was performed as in panel A. Binding of the primary antibody was detected using rabbit peroxidase conjugated anti- sheep IgG (1 in 10, 000 dilution, Pierce) followed by enhanced chemiluminscence (ECL, Amersham).