Different requirements for TLR4 and IKK2 in LPS signaling in human macrophages and SFs or HUVECs. Macrophages (A), SFs (B), or HUVECs (C) were left untreated (white bars) or treated for 30 minutes with 10 μg/mL TLR4 (black bars) or TLR2 (gray bars) neutralizing antibodies prior to addition of graded doses of chloroform-extracted LPS. After 20 hours, supernatants were collected and assayed by ELISA for TNFα and IL-6. Mean cytokine production (± SD) of triplicate cultures is shown and is representative of 5 independent experiments for macrophages and 3 independent experiments for SFs and HUVECs. (D) SFs, HUVECs, and macrophages were analyzed for the expression of TLR4 by FACS staining. A representative of 3 independent experiments is shown. HUVECs (E) and macrophages (F) were infected with adenoviruses overexpressing β-galactosidase, IKK2dn, or IκBα for 2 hours in serum-free medium. Then cells were cultured in complete medium for another day and stimulated with 1 μg/mL and 10 ng/mL LPS, respectively. After 45 minutes, cytosolic and nuclear extracts were collected and assayed for IκBα expression by Western blot (upper panel) and NF-κB DNA binding by EMSA (lower panels). A representative of 3 independent experiments from unrelated donors is shown.