DNA encoding GST-Tpl2 (1-467), FLAG-Tpl2 (1-467), GST-Tpl2 (30-467) and FLAG- Tpl2 (30-467) were transfected into IL-1R cells. After 24 hours, the cells were lysed and Tpl2 was pulled down from 1mg of cell lysate protein on Glutathione-Sepharose (for GST- Tpl2) or FLAG-coupled agarose (for FLAG-Tpl2). The protein was recovered from the beads using LDS, subjected to SDS-PAGE, transferred to a PVDF membrane and immunoblotted with the antibody. Binding of the primary antibody was detected using rabbit peroxidase conjugated antisheep IgG antibody (1:10.000 dilution, Pierce) followed by enhanced chemiluminescence (ECL, Amersham).