P nrf2

Total protein extract (30 μg of protein) or nuclear extract (10 μg of protein) from liver was resolved on an 8–10% sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred onto nitrocellulose membrane (Bio-Rad, Hercules, CA) and incubated for 2 hours in tris-buffered saline with Tween (TBST) (10 mM Tris, pH 8.0, 150 mM NaCL, and 0.1% Tween 20) containing 5% nonfat milk at room temperature. The membrane was then incubated overnight at 4°C with the following primary antibodies, SOD2 (1 : 5000, Epitomics), SOD1 (1 : 10000, Epitomics), CAT (1 : 1000, Epitomics), FOXO3a (1 : 1000, Epitomics), p-FOXO3a (ser253) (1 : 1000, Epitomics), HO-1 (1 : 2000, Epitomics), GCLC (1 : 5000, Bioworld), GCLM (1 : 1000, Epitomics), Nrf2 (1 : 500, Santa Cruz Biotechnology), p-Nrf2 (1 : 1000, Bioss), Akt and p-Akt (1 : 1000, Cell Signaling), Lamin B (1 : 500, Epitomics), and β-actin (1 : 500, Santa Cruz Biotechnology). After washing with TBST, the membrane was then incubated for 1 hour with horseradish peroxidase conjugated secondary antibody (all from Invitrogen with a dilution of 1 : 5000), and signal was detected using the SuperSignal West Pico Chemiluminescence kit on a Bio-Rad ChemiDocXRS Gel Imaging System. The band intensities were quantified by Quantity One Software version 4.6.3. Protein levels were normalized to β-actin or Lamin B as compared with the Control (set to 1).