DNA encoding wildtype GST-Tpl2 (30 – 397) was transfected into IL-1R cells. After 24 hours, the transfection medium was replaced with DMEM without or with 10 % serum and the cells were left for a further 16 hours before lysis. Tpl2 was purified by chromatography on glutathione Sepharose from 2 mg of cell lysate protein and recovered from the beads using LDS, subjected to SDS-PAGE, transferred to a PDVF membrane and immunoblotted with phospho specific antibodies that recognise Tpl2 phosphorylated at Thr 290 or Ser 62 [left hand panels] Binding of the primary antibody was detected using rabbit peroxidase conjugated anti- sheep IgG antibody (1 in 10, 000 dilution, Pierce) followed by enhanced chemiluminescence (ECL, Amersham). The right had panels show the same membranes stripped and reprobed with anti-GST.