Western Blot Analysis of Cellular Proteins

Cytosolic and nuclear extracts were prepared as previously described [72 (link),73 (link)]. The following primary antibodies were used: anti-NRF-2 (1:500, Santa Cruz Biotechnology, Heidelberg, Germany, #sc-365949), anti-Heme Oxigenase 1 (HO-1; 1:500, Santa Cruz Biotechnology, Heidelberg, Germany, #sc-136960), anti-Bax (1:500, Santa Cruz Biotechnology, #sc7480), and anti-Bcl-2 (1:500, Santa Cruz Biotechnology, #sc7382) in 1× PBS, 5% w/v nonfat dried milk, and 0.1% Tween-20 at 4 °C overnight. To ensure that blots were loaded with equal amounts of proteins, they were also probed with antibodies against the β-actin protein for cytosolic fraction (1:500; Santa Cruz Biotechnology, Heidelberg, Germany,) or lamin A/C for nuclear fraction (1:500 Sigma-Aldrich Corp., Milan, Italy). Signals were examined with an enhanced chemiluminescence (ECL) detection system reagent according to the manufacturer’s instructions (Thermo, Monza, Italy). The relative expression of the protein bands was quantified by densitometry with BIORAD ChemiDocTM XRS⁺ software and standardized to the β-actin and lamin A/C levels.