Western Blot Analysis of Cytosolic and Nuclear Proteins

Extracts of the cytosol and nucleus were prepared, as previously mentioned [82 (link),83 (link),84 (link),85 (link),86 (link)]. The following primary antibodies were used: anti-iNOS (1:500, Santa Cruz Biotechnology, #sc-7271, Dallas, TX, USA), anti-Cox-2 (1:500, Santa Cruz Biotechnology, #sc-19999), anti-FAAH (1:500, Sigma-Aldrich Corp., Milan, Italy), anti-Iκbα (1:500, Santa Cruz Biotechnology, #sc-1643), and anti-nfκb (1:500, Santa Cruz Biotechnology, #sc8414) in 1 × PBS, 5% w/v non-fat dried milk, and 0.1% Tween 20, at 4 °C overnight [87 (link),88 (link),89 (link),90 (link)]. For the cytosolic fraction, Western blots were also explored with antibody against β-actin protein (1:500, Santa Cruz Biotechnology, Dallas, TX, USA). The same methods were used for nuclear fraction with lamin A/C (1:500, Sigma-Aldrich Corp., Milan, Italy) [91 (link),92 (link)]. 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 ChemiDoc^TM XRS+ software (Hercules, CA, USA) [83 (link),87 (link),93 (link),94 (link),95 (link)].