Mal/TIRAP is dispensable for IL-1– or LPS-induced NF-κB activation or cytokine production in human macrophages. Human macrophages (A-G) or RAW cells (G) were infected for 2 hours with adenoviruses expressing β-galactosidase, Malwt, Maldn, or MalTIR in serum-free medium. Cells were cultured in complete medium for another 2 days and treated with 20 ng/mL IL-1, 0.01 ng/mL to 100 ng/mL LPS, or 20 ng/mL TNFα. 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 expression by Western blot (upper panel) and NF-κB DNA binding by EMSA (lower panel). After 20 hours of treatment with LPS, supernatants from uninfected (white bars), Adβ-gal–infected (gray bars), or AdMaldn-infected (black bars) cells were collected and assayed by ELISA for TNFα (C) and IL-6 (D). (E) Combinations of AdMyD88dn with Adβ-gal, AdMaldn, or AdMalTIR were also used to infect human macrophages, and their effect on TNFα production after treatment for 20 hours with 10 ng/mL LPS was examined. (F) Combinations of AdMalwt with Adβ-gal or AdMaldn were also used to infect human macrophages and IL-6 production in the absence of further treatment was examined after 20 hours. A 1:1 ratio means that MOI of 100 for each virus was used, whereas 1:3, 1:5, and 1:7 ratios mean that MOIs of 300, 500, and 700, respectively, were used for the higher-ratio virus. Mean cytokine production (± SD) of triplicate cultures is shown and is representative of 5 independent experiments from unrelated donors. Finally, total mRNA levels from RAW cells or human macrophages were extracted, quantified, and equal amounts subjected to RT-PCR for IFNβ and glyceraldehyd-3-phosphate dehydrogenase (GAPDH) amplification (G). A representative of 3 independent experiments is shown.