Mouse anti nr1

For Western blot analyses, the following primary antibodies were used: mouse anti-PSD-95 (1:250; BD transduction), rabbit anti-CDK5 (1:500; Santa Cruz biotechnology, Dallas TX), mouse anti-Akt (1:1000, Cell signal technology, Danvers, MA), rabbit anti-p44/42 MAPK (Erk1/2) (1:1000; Cell signal technology), mouse anti-GFAP (1:500, Sigma Aldrich, Oakville, Ontario), mouse anti-syntaxin (1:10 000, Sigma Aldrich), rabbit anti-GluR1 (1:2000, Millipore, Billrica, MA), mouse anti-GluR2 (1:2000, Millipore), mouse anti-NR1 (1:2000), rabbit anti-D2R (1:500, Millipore) and mouse anti-actin (1:10 000; Millipore). Secondary antibodies IRDye 680 Goat Anti-Rabbit IgG (1:10 000; Mandel Scientific, Guelf, Ontario) or IRDye 800 Goat Anti-Mouse (1:10 000; Mandel Scientific) were then used.
For immunochemistry analysis, mouse monoclonal anti-neuronal nuclei (NeuN) (1:250; Millipore) and mouse monoclonal anti-actin (1:5000; Millipore) were used as primary antibodies. Revelation of labeling using the Odyssey imager was performed using IRDye 800 Goat Anti-mouse IgG (1:1000) and IRDye 680 Goat Anti-mouse IgG (1:1000) secondary antibodies.

Equal amount of proteins (~15 μg for each condition) were resolved by 10% SDS-polyacrylamide gels and blotted onto PVDF membrane (Serva, Germany). The resulting blot was blocked for 1 h at room temperature using Tris-buffered saline-Tween (t-TBS) (M) Tris, 0.02; NaCl, 0.15; Tween 20, 0.1%) containing 5% skimmed milk. The membranes were then incubated overnight at 4°C with specific antibodies: rabbit phospho-SAPK/JNK (Thr 183/Tyr185) (p-JNK) 1:500 (Cell Signaling, USA) this antibody recognizes altogether the phosphorylated bands (46 and 54 kDa) of JNK1, JNK2 and JNK3, rabbit SAPK/JNK 1:1000 (Millipore, Italy) this antibody recognizes altogether the bands (46 and 54 kDa) of JNK1, JNK2 and JNK3, mouse β-actin 1:30000 (Sigma Aldrich, Italy), mouse anti NR1 1:1000 (Millipore), mouse anti NR2a and NR2b 1:1000 (Millipore), rabbit anti GluA1 and GluA2 1:1000 (Millipore).
After 50 min of wash in t-TBS, the blots were incubated for 1 h at room temperature with peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibodies (UCS Diagnostic).
After 50 min of washes in t-TBS bands immunoreactivity was detected by enhanced chemiluminescence (ECL; WESTAR, Cyanagen, Italy). In all experiments where an evaluation of phosphorylated proteins was required, pan antibodies were applied on the same membranes after stripping procedure (stripping buffer from SignaGen, USA).