Westar nova 2011

DRG or sciatic samples were homogenized in lysis buffer (1% Triton, 20 mM Tris pH 7.6, 1 mM EDTA, 150 mM NaCl, 1 mM NaF, 1 mM NaVO3) with protein inhibitor cocktail (Sigma) followed by total protein quantification. Proteins were resolved in 12% SDS-PAGE gel and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was then blocked with 5% non-fat milk in TBS-T solution for 1 h, followed by incubation with anti-GAP-43 (sciatic sample, Santa Cruz, RRID:AB_640874), anti-ATF-3 (DRG sample, Santa Cruz, RRID:AB_2258513) or anti-β-actin/actin primary antibody overnight at 4°C (1:1000, Santa Cruz, RRID:AB_2223228 /Millipore, RRID:AB_2223041). On the next day, anti-rabbit secondary antibody was incubated for 1 h and chemiluminescence was measured using a ChemiDoc XRS+ imaging system (Bio-Rad) after development in chemiluminescence reagent (Westar Nova 2011, CYANAGEN). Bands intensity was determined using ImageJ software (NIH, United States).

Cells were lysed in RIPA buffer, separated by SDS–PAGE and transferred to nitrocellulose membranes following standard protocols. The membranes were incubated overnight with the following primary antibodies: anti-phospho-Src (Tyr416) (2101, 1:1000), anti-phospho-AKT (Ser473) (9271; 1:1000) and anti-phospho-SMAD3 (Ser423/425) (9520, 1:1000) (Cell Signalling Technology Boston, USA); anti–Tubulin (TU-02, 1:1000), and anti–CDK4 (c-22; 1:1000) (Santa Cruz Biotechnology, Inc., CA, USA). Blots were incubated with HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Immunoreactivity was detected by Enhanced Chemiluminescence reaction (WESTAR NOVA 2011, Cyanagen, Bologna, Italy) following the manufacturer’s instructions.

Western blot analysis of mGlu5 receptors was carried out in the hippocampus, prefrontal cortex, corpus striatum, olfactory bulb, hypothalamus, and cerebellum of four CD1 mice randomly selected from the groups used for the assessment of PI hydrolysis. An aliquot of tissue extract was added with protease inhibitors cocktail (Sigma-Aldrich, Cat. # 2714) and after protein determination, protein lysates (40 μg) were separated by SDS-PAGE electrophoresis and blotted onto nitrocellulose. The upper part of the membrane was probed with polyclonal anti-mGlu5 receptor antibody (Millipore, CA, United States, Cat. # AB5675, 1:3,000 dilution), and the lower part of the membrane was probed with monoclonal anti-β-actin antibody (Sigma-Aldrich, Cat. # A2228, 1:10,000 dilution). Immunoreactive bands were visualized by enhanced chemilumiscence (Westar, Nova 2011, Cyanagen, Bologna, Italy) using horseradish peroxidase-conjugated secondary antibodies. Densitometric analysis of the immunoreactive bands was performed by Image J (NIH, Bethesda, MD, United States).