Fig. 6. TACE phosphorylation at Thr735 is mediated by the TPL2-MKK1/2-ERK1/2 pathway. (A) The C-terminal tail of TACE [TACE(693-827)] was expressed in bacteria as a GST-fusion, purified and phosphorylated with or without ERK2, as described (Morton et al., 2006). The indicated amounts (ng) of TACE(693-827) were spotted onto nitrocellulose and probed with an antibody that recognises TACE phosphorylated at Thr735 (pTACE Thr735). (B) RAW cells were pre-treated for 60 minutes without (–) or with (+) 10 µM U0126 (U0) or 2 µM PD 184352 (PD) and then either left untreated or were stimulated for 45 minutes with 100 ng/ml LPS. Immunoblotting was carried out as in Fig. 1 using an antibody that recognises ERK1/2 phosphorylated at the TEY motif (pERK), one that recognises TACE phosphorylated at Thr735 or an antibody that recognises the phosphorylated and unphosphorylated forms of TACE equally well (TACE). (C) BMDM from wild-type (WT) or TPL2-deficient (TPL2–/–) mice were either left untreated or were stimulated with 100 ng/ml LPS for the times (minutes) indicated. Immunoblotting was performed as in B.