FIGURE 3. IKK- regulates hypoxia-induced HIF-1 and target genes in mouse macrophages.

a, BMDM from IKK^F/F (IKK-^+/+) or IKK (IKK-^-/-) mice were incubated with DFX for 4 h. Expression of the indicated proteins was analysed by immunoblotting for nuclear (NE) and cytosolic (CE) extracts. b, BMDM were obtained as above and cultured under normoxia or hypoxia (0.5% O₂ for 4 h). Protein expression was analysed by immunoblotting. c, BMDM were treated as above and mRNA expression was analysed by quantitative RT–PCR. Light grey bars, normoxia; dark grey bars, hypoxia. Results are means and s.e.m. for three separate experiments performed in triplicate. *, P < 0.05 versus normoxic IKK^+/+ cells; †, P < 0.05 versus hypoxic IKK^+/+ cells. PGK, phosphoglucokinase; iNOS, inducible nitric oxide synthase. d, RAW264.7 macrophages were cultured in the absence or presence of LPS under the indicated O₂ tensions for 2 h. Protein expression was analysed by immunoblotting. e, Murine embryonic fibroblasts from IKK^+/+ or IKK^-/- embryos were transfected with a luciferase reporter gene driven by the Hif1a promoter. After 36 h the cells were incubated for 3 h with DFX. Light grey bars, control; dark grey bars, DFX. Results are means and s.e.m. for three separate experiments performed in triplicate.