2.5. Western Blot Analysis

Cells were lysed with a hypotonic buffer solution containing 20 mM Tris (pH 7.4), 10 mM NaCl, 3 mM MgCl2 and a protease inhibitor mixture. After addition of 10% Triton-X 100, cell lysates were centrifuged at 650× g for 10 min at 4 °C and supernatants were collected as cytosolic fractions. Remaining pellets were resuspended in cell extraction buffer [100 mM Tris (pH 7.4), 1% Triton X-100, 10% glycerol and 0.1% SDS] containing protease inhibitor mixture. Homogenates were then centrifuged at 14,000× g for 20 min at 4 °C and supernatants were collected as nuclear fractions. The levels of nuclear factor erythroid 2-related factor 2 (Nrf2) quantified in nuclear fraction. The filters were blocked with 1 × PBS, 5% (w/v) nonfat dried milk (PM) for 40 min at room temperature and subsequently probed with specific Nrf2 antibody (Novus biologicals, Centennial, CO, USA) in 1 × PBS, 5% w/v nonfat dried milk, 0.1% Tween-20 (PMT) at 4 °C, overnight. Membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson Immuno Research, West Grove, PA, USA) at room temperature for 1 h. Laminin protein were used as internal standard for nuclear extracts. Signals were detected with enhanced chemiluminescence detection system reagent according to manufacturer’s instructions (Super-Signal West Pico Chemiluminescent Substrate, Pierce). The relative expression of the protein bands was quantified by densitometry with Bio-Rad (Bio-Rad, Milan, Italy) ChemiDoc XRS software and standardized to lamin levels. A preparation of commercially, molecular weight markers made of proteins of molecular weight 10–250 kDa was used to define molecular weight positions and as reference concentrations for each molecular weight.