Figure 2

PI3K is involved in shear-induced Nrf2 nuclear translocation. (A) PI3K is involved in shear-induced translocation of Nrf2 into nuclei. HUVECs were pretreated with LY294002, a PI3K inhibitor (50 μM), for 60 min and then kept as static controls or exposed to laminar shear stress (12 dyne/cm²) in the presence of LY294002 for 120 min. Nuclear extracts were prepared and subjected to Western blotting with an anti-Nrf2 antibody. The cytoplasmic fractions were subjected to Western blotting with an anti-tubulin (internal control) antibody. (B) PKC is involved in Nrf2 activation. HUVECs were pretreated with calphostin C, a PKC inhibitor (200 nM), or DMSO as a negative control for 30 min, and then exposed to laminar shear stress (12 dyne/cm²) or kept in a static condition for 2 h. After treatment, nuclear extracts were prepared and subjected to SDS-PAGE and immunoblotted with anti-Nrf2 and anti-nucleolin (internal control) antibodies. Similar results were obtained from repeated experiments. (C) p38 is not involved in shear-induced Nrf2 translocation. HUVECs were pretreated with SB203580, a p38 inhibitor (10 μM), for 30 min, and then kept as a static control or exposed to shear stress in the presence of inhibitors for 60 min. Nuclear extracts were prepared and subjected to Western blotting with anti-Nrf2 and anti-αC23 (internal control) antibodies. Similar results were obtained from repeated experiments. (D) JNK is not involved in shear-induced Nrf2 translocation. HUVECs were pretreated with SP600125, a JNK inhibitor (10 μM), for 60 min, and then kept as a static control or exposed to shear stress in the presence of the inhibitor for 60 min. Nuclear extracts were prepared and subjected to Western blotting with anti-Nrf2 and anti-αC23 (internal control) antibodies. Similar results were obtained from repeated experiments.