PI3K/Akt pathway is involved in activation of HIF-1α in A204 and A673 cell lines. Cells were pretreated with 0 or 30 μmol/L of LY294002 for 1 hour and then incubated for 24 hours or 48 hours either in normoxia (N) or hypoxia (H). A. Protein expression levels of HIF-1α and β-actin were analyzed by western blotting. B. DNA binding activity of Hif-1α in nuclear extracts was assessed by EMSA. Specific HIF-1 DNA binding was confirmed by using a radioactive labeled mutated (m) probe, in which the HIF-1α consensus binding site is inactivated, and by competition with unlabelled consensus (c) and mutant (c/m) DNA probes in 100 fold excess.