Figure 4.

Effects of 2ME2 on pro‐death or pro‐survival gene expression after ischemic challenge. A. and B. Time course expression of VEGF and Nix in the primary neurons and ischemic hemispheres. Total lysates were prepared from ischemic hemispheres (A) and primary neurons (B). The levels of VEGF, Nix and HIF‐1α were determined by performing immunoblotting with anti‐VEGF antibodies, anti‐Nix antibody and anti‐HIF‐1α antibody, using actin as an internal control. C–F. Total lysates of the primary neurons were obtained at various time points following 2ME2 treatment (C,F), 2ME2 treatment 0.5 h (D) or 8 h (E) after OGD challenge. VEGF, Nix or HIF‐1α expression was detected by performing immunoblotting with anti‐VEGF antibodies, anti‐Nix antibody, anti‐HIF‐1α antibody, anti‐NF‐κB p65 antibodies and anti‐c‐fos antibodies, using actin as an internal control. All of the experiments were quantified in three independent experiments and were expressed as a percentage of the levels in the naive‐controls. Statistical analysis was carried out using the one‐way ANOVA with appropriate post hoc tests; **P < 0.01 vs. the naive‐control group. Abbreviations: 2ME2 = 2‐methoxyestradiol; VEGF = vascular endothelial growth factor; Nix = Nip‐like protein X; HIF‐1α = hypoxia‐inducible factor‐1‐alpha; OGD = oxygen–glucose deprivation; NF‐κB = Nuclear factor‐kB; MCAO = middle cerebral artery occlusion.