Hypoxia does not activate NF-κB. Macrophages were incubated under normoxic (N) or hypoxic (H) conditions for up to 30 min. and nuclear levels of NF-κB determined by EMSA (8A). Hypoxia does not activate NF-κB activity above basal normoxic levels. LPS (1 µg/ml for 30 min.) strongly induces NF-κB activation (n = 4, P < 0.01). A representative EMSA is shown including a competition assay (8B). Lane 3 contains 4 µg of protein from LPS-stimulated macrophages. Lane 2 as 3 with the addition of 100× excess cold non-competitor. Lane 1, 100 × excess cold NF-κB competitor. A supershift assay further defines this LPS-induced band (8C). Lane 4, nuclear extracts from LPS-stimulated macrophages (as Lane 1). Subsequent lanes with addition of 2 µl control rabbit serum (Lane 5), p50 antibody (Lane 6) revealing two distinct supershifted bands) or p65 antibody (Lane 7 revealing one supershifted band).