Figure 9

Influence of hypoxia on P ₂ promoter binding by c-myc promoter binding protein in response to 1 nM, 5 mM, or 25 mM glucose as determined by electrophoretic mobility shift assay. A) A 50 base pair c-myc P₂ promoter was ³²P-labeled and incubated with nuclear extract (NE). Cold competitor MP2 (150 nM) (Lane 9) or cold nonspecific oligonucleotide BEE-1 (150 nM) (Lane 10) was added prior to addition of the probe. Note that MBP-1 binding to the probe with 1 nM glucose was similar to the 0 h after 4 and 24 h of hypoxia. This is in contrast to normoxia data which showed an early increase in binding compared to baseline. Binding was greatly reduced in the 5 mM and 25 mM groups after 4 and 24 h of hypoxia compared to normoxia. B) An α-enolase antibody was added to a reaction containing the nuclear extract and ³²P-labeled P₂ promoter (lane 3) causing a supershift of the DNA-protein complex. This supershift was competed off with MP2 cold competitor (lane 4), but not with the cold nonspecific competitor BEE-1 (lane 5). No supershift occurred with addition of rabbit IgG to the reaction in place of antibody (lane 6). Normoxia figure was adapted and previously published [26].