Generation of cell lines with stable expression of CYLD mutants

U2OS/NOD2 CYLD KO cells were retrovirally transduced with CYLD-encoding virus particles. Full-length WT CYLD was cloned into the retroviral pBabe-puro vector (Morgenstern and Land, 1990). CYLD mutations (L475P, S418A, S568A, S418A/S568A, and C601) were generated by site-directed mutagenesis and were verified by sequencing. Retroviral particles were generated by transfection of pBabe constructs (10 μg) into Phoenix-AMPHO cells in 10 cm plates and culturing for 3 days. Supernatant was collected and filtered through a 0.45 μm filter. Final concentrations of 5% w/v PEG-8000 and 150 mM NaCl were added to the supernatant and incubated with rotation at 4°C overnight. Virus particles were collected by centrifugation at 3500 g for 15 min and pellets resuspended in sterile PBS. U2OS/NOD2 CYLD KO cells were infected with the concentrated retroviral particles (1 – 4 μl) in the presence of 6-10 μg/ml polybrene (Sigma-Aldrich) over night and selected with 1 μg/ml puromycin (Invivogen) for one week.