Bladder and Testes Protein Extraction

Cytosolic and nuclear extracts were prepared from the bladder and testes as previously described [69 (link),70 (link)]. The following primary antibodies were used: anti-NRF-2 (1:500, Santa Cruz Biotechnology, Heidelberg, Germany, #sc-365949), anti-caspase 3 (1:500, Santa Cruz Biotechnology, Heidelberg, Germany, #sc-7272), anti-heme oxygenase 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). These were mixed 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 β-actin protein for cytosolic fraction (1:500; Santa Cruz Biotechnology Heidelberg, Germany) or lamin A/C for nuclear fraction (1:500 Sigma-Aldrich, 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 Bio-Rad ChemiDoc^TM XRS⁺ software (Version 6.0.1, Milan, Italy) and standardized to the β-actin and lamin A/C levels [71 (link),72 (link),73 (link),74 (link),75 (link)].