Western Blot Analysis of Cytosolic and Nuclear Proteins

Cytosolic and nuclear extracts were prepared as previously described [69 (link),70 (link)]. The following primary antibodies were used for cytosolic fraction: anti-IκBα (1:500, Santa Cruz Biotechnology, #sc1643), anti-Bax (1:500, Santa Cruz Biotechnology, Dallas, TX, USA, #sc7480), anti-Bcl-2 (1:500, Santa Cruz Biotechnology, #sc7382), cleaved-caspase 3 (1:500, Cell Signaling, Danvers, MA, USA), and β-actin (1:1.000; Santa Cruz Biotechnology, #sc8432). The following primary antibodies were used for nuclear fraction: anti-NF-κB p65 (1:500, Santa Cruz Biotechnology, #sc8008), and lamin A/C antibody (1:5000; Sigma-Aldrich, St. Louis, MO, USA). Protein expression was quantified by densitometry with BIORAD ChemiDocTM XRS+software and standardized to β-actin and lamin A/C levels [71 (link)].