Ecl detection system reagent

Extracts of the cytosol and nucleus were prepared, as previously mentioned [62 (link),63 (link),64 (link),65 (link),66 (link)]. The following primary antibodies were used: anti-α-sma antibody (1:250, Santa Cruz Biotechnology (SCB), Dallas, TX, USA), anti-Iκbα (1:500, SCB, #sc-1643), anti-ICAM (1:500, SCB), anti-pselectin (1:500, SCB), anti-TGFβ (1:500, SCB) and anti-nfκb (1:500, SCB, #sc8414) in 1× PBS, 5% w/v non-fat dried milk, and 0.1% Tween 20, at 4 °C overnight [67 (link),68 (link),69 (link)]. Western blots were further investigated for the cytosolic fraction using an anti-β-actin protein antibody (1:500, SCB, Dallas, TX, USA). For nuclear fraction with lamin A/C (1:500, Sigma-Aldrich Corp., Milan, Italy), the same techniques were applied [70 (link),71 (link)]. According to the manufacturer’s instructions, an enhanced chemiluminescence (ECL) detection system reagent (Thermo, Monza, Italy) was used to examine the signals. Using densitometry and the BIORAD Chemi-DocTM XRS+ software (Bio-Rad, Milan, Italy), the relative expression of the protein bands was measured [72 (link),73 (link)].

Western blot analysis was performed as described [83 (link)].
Briefly, after protein extraction from brain tissues, cytosolic lysates were used for the detection of BDNF and NT-3.
After SDS-PAGE, the proteins present in the polyacrylamide gel are transferred to the PVDF membrane. Membranes were incubated at 4 °C overnight with each of the following primary antibodies: anti-BDNF (1:500; sc-20981; Santa Cruz Biotechnology, Dallas, TX, USA), anti-NT-3 (1:500; sc-547; Santa Cruz Biotechnology, Dallas, TX, USA) dissolved in a PMT solution consisting of 1× phosphate buffer saline (PBS), 5% w/v nonfat dried milk powder and 0.1% Tween-20. Thereafter, the membranes were washed and incubated with secondary antibody (1:1000, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature.
To confirm that the samples used contained a uniform concentration of protein lysates, they were incubated in the same way, with primary anti-β-actin antibody (1:500; sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA). Signals were exposed with chemiluminescence (ECL) detection system reagent according to the manufacturer’s instructions (Thermo, Waltham, MA, USA). The relative expression of the protein bands was quantified by densitometry and standardized to β-actin levels, as an internal control.

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)].