CYLD was immunoprecipitated from HEK293-IL1R total cell extracts (1.5 mg) using various amounts of anti-CYLD S413D before being immunoblotted with anti-CYLD (Cell Signaling

4495S). The cell supernatant obtained after immunoprecipitations (20 μg) were immunoblotted with anti-CYLD (S413D). Binding of the primary antibody was detected using goat peroxidase conjugated anti- rabbit IgG antibody (1:10000 dilution) or rabbit peroxidase conjugated anti-sheep IgG antibody, respectively followed by enhanced chemiluminescence (ECL, Amersham).