Figure 3. Comparison of triggered NF-κB activation and cytokine production in Cyld^–/– and wild-type cells.

(A) Peritoneal macrophages were stimulated with TNF-α (500 U/ml), CD40 agonist Ab (1 μg/ml), LPS (2.5 μg/ml), or Pam₃CSK₄ (150 ng/ml). Cellular extracts were prepared from cells stimulated for the indicated intervals and analyzed by EMSA for NF-κB binding activity. Quantification of NF-κB activity is represented as fold change compared with unstimulated cells (set at 1.0). (B) Peritoneal macrophages were stimulated with LPS (2.5 μg/ml), Pam₃CSK₄ (150 ng/ml), or CD40 agonist Ab (5 μg/ml) for 24 hours. IFN-γ (10 ng/ml) was added to all stimulation conditions, and the production of IL-6 and TNF-α into culture supernatants was assessed by ELISA. Med, medium. *P < 0.005, Cyld^–/– versus wild-type. (C) CYLD overexpression suppresses NF-κB activation by CD40, EDAR, and RANK, but not by TNF-α. 293M cells were transfected with an NF-κB reporter construct either in combination with a CYLD-bearing plasmid or with empty vector as a control. Cells were stimulated with TNF-α, and luciferase activity was measured. To activate other TNFR pathways, vectors bearing CD40, EDAR, and RANK were cotransfected into 293M cells. This led to receptor overexpression and ligand-independent NF-κB activation caused by spontaneous receptor trimerization. Quantitation of NF-κB activity in the presence of CYLD is indicated as fold suppression. *P < 0.005, Cyld^–/– versus vector.