Fig. 4. Expression of IB and IKK-2dn inhibits CD40L-induced VEGF release by macrophages. (A) M-CSF-differentiated macrophages were cultured for 24 hours in 30 mm² wells with 5x10⁵ CD40L-transfected (CD40L+) or control (MOCK) cells. After 24 hours, supernatants were collected and assayed by ELISA for VEGF. In parallel experiments, macrophages were left uninfected, or infected for 2 hours in serum-free medium with Advß-gal, AdvIKK-2dn and AdvIB, at m.o.i. 100:1. Cells were cultured for a further 2 days and then stimulated with 1 µg/ml sCD40L. After 45 minutes, cytosolic and nuclear extracts were obtained and examined for the presence of IKK-2dn (B) and IB (C) by western blotting. A representative of three independent experiments is shown. Equal amounts of protein were loaded, as determined by re-probing for -tubulin (not shown). For measurement of VEGF production, macrophages were left uninfected, or infected as described above and after 2 days stimulated with CD40L-transfected (CD40L+) or (MOCK) cells as control. After 24 hours, supernatants were collected and assayed for VEGF (D). Mean cytokine production±s.d. of triplicate cultures is shown and is representative of three independent experiments: ^**P<0.01, ^***P<0.001 versus response with control adenovirus-infected and uninfected cells.