Western Blot Analysis of Spinal Cord and Disc Tissues

Spinal cord and lumbar disc 5–6 tissues were homogenized, and Western blots were performed as already described [79 (link),80 (link),81 (link)]. The samples were stored at −80 °C for further analysis.
Specific primary antibodies against Aggrecan (sc-166951, Santa Cruz Biotechnology), NGF (MA5-32067, Thermo Fisher, Milan, Italy), trkA (JJ084-04, Thermo Fisher), WNT3a (sc-80457, Santa Cruz Biotechnology), anti-FZ8 (Bioworld Technology, St. Louis Park, MN, USA), anti–β-catenin (610153, BD Biosciences), anti-active β-catenin (05-665, Millipore, Milan, Italy), anti-NFkB (Santa Cruz Biotechnology, sc-8008), anti-NOS2 (Santa Cruz Biotechnology, sc-7271), or anti-COX-2 (Santa Cruz Biotechnology, sc-376861) were mixed in a 5% w/v nonfat dried milk solution and incubated with the membranes at 4 °C overnight. Afterwards, the blots were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibodies or peroxidase-conjugated goat anti-rabbit IgG (Jackson Immuno Research, West Grove, PA, USA) for 1 h at room temperature [82 (link)]. The membranes were also incubated with antibodies against β-actin or lamin A/C (Santa Cruz Biotechnology) to verify that equal amounts of protein were loaded [83 (link)]. Images of the blot signals were imported into an analysis software (v2003, Image Quant TL, Milan, Italy) [84 (link)].