4.9. Western Blot Analysis of Activation of Nrf2 and Stress-Response Pathway

Western blot analysis was used to demonstrate the activation of Nrf2 and its downstream regulated genes, including HO-1 and NQO1 [20]. Protein extracts were done as described previously [46]. Mouse monoclonal anti-Nrf2 antibody (A-10; 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) was used to determine human ~60 kDa forms of Nrf2. Rabbit polyclonal anti-HO-1 antibody (1:1000; Thermo Fisher Scientific, Waltham, MA, USA) for detection of HO-1 and rabbit polyclonal anti-NQO1 antibody (1:1000; Thermo Fisher Scientific, Waltham, MA, USA) for the detection of HO-1 and NQO1, respectively. Polyclonal anti-HSP90 antibody and polyclonal anti-HSP70 antibody (both 1:2000; Cell Signaling Technology, Danvers, MA, USA) was used for detection of reference protein. Goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP secondary antibody (1:2000 or 1:10,000; Dako, Glostrup, Denmark) in combination with an enhanced chemiluminiscence (ECL; Amersham, Little Chalfont, UK) was used for signal detection.