Fig. 2. Overexpression of IB in M-CSF-differentiated human macrophages inhibits LPS-induced NF-B activation and VEGF production. Macrophages were either left uninfected or were infected for 2 hours in serum-free medium with control adenovirus, or an adenovirus expressing IB. Cells were cultured in 5% FCS for 2 days and subsequently stimulated with 10 ng/ml LPS for 45 minutes. Cytosolic and nuclear extracts were then collected and examined for cytosolic levels of IB (A) and -tubulin (B) by western blot analysis or for NF-B translocation by EMSA (C). A representative of three independent experiments is shown. For VEGF production, macrophages were left uninfected, or infected for 2 hours in serum-free medium with control adenovirus or with an adenovirus-expressing IB. After 2 days of further culture, cells were stimulated with 10 ng/ml LPS for 24 hours. Supernatants were collected and assayed by ELISA (D). Data are means±s.d. of culture triplicates, and are representative of ten different experiments: ^***P<0.001 versus response with control adenovirus-infected and uninfected cells.