Fig. 3. Expression of IKK-2dn in human macrophages does not inhibit LPS-induced NF-B activation but partially decreases VEGF production. Macrophages were either left uninfected, or were infected for 2 hours in serum-free medium with control adenovirus, or adenoviruses expressing IKK-1dn and IKK-2dn. Cells were cultured in 5% FCS for 2 days and stimulated with 10 ng/ml LPS for 45 minutes. Cytosolic and nuclear extracts were then collected and examined for cytosolic levels of IKK-2 (A), IB (B) and -tubulin (C) by western blot analysis or for NF-B translocation by EMSA (D). A representative of three independent experiments is shown. For VEGF production, macrophages were left uninfected, or were infected for 2 hours in serum-free medium with control adenovirus or with an adenovirus expressing IKK-2dn. After 2 days of further culture, cells were stimulated with 10 ng/ml LPS for 24 hours (E). Data are expressed as means±s.d. of triplicates and are representative of ten different experiments: ^*P<0.05 versus response with control adenovirus-infected and uninfected cells.