Figure 5

Shear stress increases the antioxidant response element (ARE)-binding activity of Nrf2 through reactive oxygen species (ROS). HUVECs were kept in a static condition (Control), exposed to shear stress (SS) of 12 dyne/cm² for 2 h, incubated with H₂O₂ (200 μM) for 2 h (H₂O₂), or pretreated with NAC (10 mM) (NAC) and then incubated with H₂O₂ (200 μM) for 2 h (NAC+H₂O₂). Before applying the shear stress, HUVECs were pretreated with NAC (10 mM) for 60 min and then exposed to shear stress for 2 h (NAC+SS). Total nuclear extracts were prepared and analyzed by EMSA using a biotin-labeled oligonucleotide probe containing Nrf2 consensus binding sites corresponding to the HO-1 promoter region (5'-GGG ACT GGT GAC TCA GCA AAA TCT-3', within the promoter region lying about 4 kb upstream of the human HO-1 gene). The specificity of the Nrf2 binding was assessed by preincubating nuclear extracts with the biotin-labeled oligonucleotide probe in the presence of 100× unlabeled oligonucleotide probe to compete for Nrf2 binding (Competition). EMSA performed on the nuclear extracts preincubated with an Nrf2 antibody (SS+Nrf2 Ab) was also included. Results are representative of duplicate experiments with similar results.