Nuclear Protein Extraction and Western Blot Analysis

Western blots were performed as described in our previous studies [55 (link)]. Pancreas and colon tissue from each mouse were suspended in an extraction’s buffer containing 0.15 µM pepstatin A, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium orthovanadate and 20 µM leupeptin, homogenized at the highest setting for 2 min and centrifuged at 1000× g for 10 min at 4 °C. Supernatants contained the cytosolic fractions, while the pellets represented the nuclear ones. Pellets were resuspended in a second buffer containing 150 mM sodium chloride (NaCl), 1% Triton X-100, 1 mM ethylene glycol tetraacetic acid (EGTA), 10 mM tris–chloridric acid (HCl) pH 7.4, 0.2 mM PMSF, 1 mM Ethylenediaminetetraacetic acid (EDTA), 0.2 mM sodium orthovanadate and 20 µm leupeptin. After centrifugation at 4 °C for 30 min, the nuclear proteins containing the supernatants were stored at −80 °C for further analysis. Specific primary antibody anti-Nrf2 (1:1000, Santa Cruz Biotechnology) or anti-HO-1 (1:1000; Santa Cruz Biotechnology) was mixed in 1× PBS, 5% w/v nonfat dried milk and 0.1% Tween-20 and incubated at 4 °C overnight. After, blots were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson Immuno Research) for 1 h at room temperature. To verify the equal amounts of protein, membranes were also incubated with the antibody against laminin (1:1000; Santa Cruz Biotechnology) and β-actin (1: 1000; Santa Cruz Biotechnology). Signals were identified with enhanced chemiluminescence (ECL) detection system reagent and the relative expression of the protein bands was measured by densitometry with BIORAD ChemiDocTM XRS+software (Bio-rad, Milan, Italy). A representation of blot signals was imported to analysis software (Image Quant TL, v2003).