Western blot analysis was performed on spinal cord and brain samples with the protocol that we previously described [61 (link),62 (link),63 (link)]. Specific primary antibody: anti-P2X7R (1:1000, Cell Signaling Technology, Danvers, MA, USA); anti-NGF (1:500, Santa Cruz Biotechnology (SCB), #sc-32300), anti-c-FOS (1:500, SCB, sc-166940), anti-NLRP3 (1:1000, Cell Signaling Technology), anti-ASC (1:1000, SCB, #sc 271054), anti-Casp-1 (1:1000, Cell Signaling Technology) were mixed in 1× PBS, 5% w/v nonfat dried milk, 0.1% Tween-20, and incubated at 4 °C, overnight. After, blots were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson Immuno Research, West Grove, PA, USA) for 1 h at room temperature. To assess whether blots were loaded with equal volumes of protein lysates, we probed them with a mouse monoclonal β-actin (1:5000; SCB, #sc8432). Signals were detected with an enhanced chemiluminescence detection system reagent according to manufacturer’s instructions (Super-Signal West Pico Chemiluminescent Substrate, Pierce, Altrincham, UK) [64 (link)]. Protein expression was quantified by densitometry with BIORAD ChemiDocTM XRS+ software and standardized to β-actin levels. Images of blot signals were imported to analysis software (Image Quant TL, v2003) [65 (link)].
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