Lumbar spinal cord tissues were homogenized and Western blots were performed as already described [54,55]. A specific primary antibody, such as WNT3a (sc-80457), anti-FZ1, anti-FZ8, anti–β-catenin, anti-active β-catenin, anti-pNR2B, anti- pPKCγ, anti-p-CREB, anti-NFkB (sc-8008), anti-NOS2 (sc-7271), or anti-COX-2 (sc-376861), was mixed in 5% w/v nonfat dried milk solution and was incubated at 4 °C overnight. Afterwards, blots were incubated with peroxidase-conjugated bovine antimouse IgG secondary antibody or peroxidase conjugated goat anti-rabbit IgG for 1 h at room temperature [56,57]. To verify the equal amounts of protein, membranes were also incubated with the antibody against β-actin or lamin A/C. Signals were detected with enhanced chemiluminescence detection system reagent (Super-SignalWest Pico Chemiluminescent Substrate, Pierce, Monza, Italy) [58,59,60]. The relative expression of the protein bands was quantified by densitometry with Bio-Rad ChemiDoc XRS software (Bio-Rad, Milan, Italy) and standardized to β-actin or lamin A/C levels. Images of blot signals were imported to analysis software (v2003, Image Quant TL, Amersham Biosciences, Freiburg, Germany) [59]. The western blots analyses are representative of 3 different gels made by dividing the number of samples obtained from animals for each experimental group in different days [61,62].

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