Antibody against β actin protein

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)].

Extracts of the cytosol and nucleus were prepared, as previously mentioned [84 (link),87 (link),88 (link),89 (link),90 (link)]. The following primary antibodies were used: anti-Bax (1:500, Santa Cruz Biotechnology, #sc7480), anti-Bcl-2 (1:500, Santa Cruz Biotechnology, #sc7382), 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 [91 (link),92 (link),93 (link),94 (link)]. For the cytosolic fraction, Western blots were also probed 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) [95 (link),96 (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 ChemiDocTM XRS+ software [84 (link),91 (link),97 (link),98 (link),99 (link)].

Western blot analysis was performed on the lumbar portion of the spinal cord; samples were homogenized in lysis buffer as described previously [24 (link)]. The membranes were probed with specific Abs: anti-glial fibrillary acidic protein (GFAP) (1:1000; Santa Cruz) and anti-ionized calcium binding adaptor molecule 1 (Iba1) (1:1000; Santa Cruz) in 1 × phosphate buffered saline (PBS), 5% w/v nonfat dried milk, and 0.1% Tween-20 at 4 °C overnight. To ascertain that the blots were loaded with equal amounts of proteins, they were also incubated in the presence of the antibody against β-actin protein (cytosolic fraction 1:500; Santa Cruz Biotechnology). Signals were detected with an enhanced chemiluminescence (ECL) detection system reagent according to the manufacturer’s instructions (Thermo, Rockford, IL, USA). The relative expression of protein bands was quantified by densitometry with Bio-Rad ChemiDocTM XRS+ software and standardized to β-actin. Images of blot signals (8 bit/600 dpi resolution) were imported to analysis software (Image Quant TL, v2003). The blot was stripped with glycine 2% and re-probed several times to optimize detection of proteins and visualize other proteins without the need for multiple gels and transfers.