MyD88dn inhibits IL-1– but not LPS-induced NF-κB activation or cytokine production (for LPS) in human macrophages. Macrophages were generated from peripheral blood monocytes after 2 days of culture with 100 ng/mL M-CSF. Marcophages were infected for 2 hours with adenoviruses overexpressing β-galactosidase or MyD88dn in serum-free medium. Then cells were cultured in complete medium for another 2 days and treated with 20 ng/mL IL-1, 20 ng/mL TNFα, or 0.01 ng/mL to 100 ng/mL LPS. For Western blot and EMSA, 10 ng/mL LPS was used. (A) The expression of MyD88dn at different MOIs was examined by Western blot. (B) After 45 minutes of treatment, cytosolic and nuclear extracts were collected and assayed for IκBα by Western blot (upper panel) and NF-κB DNA binding by EMSA (lower panels). (C) After 20 hours of treatment with LPS, cytosolic extracts were collected and assayed for IRAK degradation by Western blot. After 20 hours of treatment with LPS, supernatants from uninfected (white bars), Adβ-gal–infected (gray bars), or AdMyD88dn-infected (black bars) cells were collected and assayed by ELISA for TNFα (D) and IL-6 (E). Mean cytokine production (± SD) of triplicate cultures is shown and is representative of 5 independent experiments from unrelated donors.