Changes in α-enolase protein expression after hypoxia in response to 1 nM, 5 mM, or 25 mM glucose. A) Western blots of α-enolase and β-actin (loading control) from total cell lysates. B) Histogram of α-enolase expression after 4, 6, 12, 24, or 48 h of hypoxia. Bars represent mean ± SEM expressed as a ratio to 0 h from 3 separate experiments. * denotes a significant (p < 0.05) increase in the level of expression.. Note greater induction of α-enolase expression after 48 h of hypoxia at all glucose concetrations compared to normoxia, especially with 1 nM glucose. Normoxia data was previously published . C) Tyrosine phosphorylation of α-enolase after 6 h of hypoxia or normoxia with 1 nM, 5 mM, or 25 mM glucose. Total lysates were immunoprecipitated with α-enolase and immunoblotted with phosphotyrosine. Efficiency and specificity of the immunoprecipitations were verified by equal immunoprecipitation of α-enolase and an absence of α-enolase in samples immunoprecipitated with goat IgG. Note increased tyrosine phosphorylation of α-enolase in response to 1 mM or 5 mM glucose with hypoxia or normoxia.