Hypoxia activates AP-1. Macrophages were incubated under normoxic (N) or hypoxic (H) conditions for up to 30 min. and nuclear levels of AP-1 determined by electromobility gel-shift assay (EMSA). Compared to normoxic controls, (6A) hypoxia significantly increased AP-1 activity at 15 and 30 min. respectively as measured by densitometry (A) (n = 4, P = 0.02). LPS (1 µg/ml) for 30 min. does not induce AP-1 activation. PMA (100 nM), a known potent upregulator of AP-1 is used as a positive control. A representative EMSA is shown (6B). The specificity and nature of the observed AP-1 band is addressed by super-shift assay (6C). Lane 1 contains 4 µg of nuclear protein from hypoxic macrophages incubated with end-labelled AP-1 oligonucleotide probe only. Subsequent lanes are as Lane 1 with the addition of 100× excess cold AP-1 competitor (Lane 2), 100X excess cold non-competitor (Lane 3), 2 µl rabbit serum (Lane 4 with non-specific band), c-fos antibody (Lane 5) or c-jun antibody (Lane 6 with super-shifted band).