Immortalized embryonic fibroblasts from wild type mice (WT) and TAB1 -/- mice were serum starved for six hours, then stimulated for the times indicated with 10 ng/ml TNFalpha. The TAK1 complex was immunoprecipitated from 0.5 mg cell extract protein using a total TAK1 antibody (Santa Cruz). The immunocomplex was denatured in SDS, separated by SDS-PAGE, transferred to nitrocellulose membrane and blotted with the appropriate antibody. 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)