Anti p camkii

All rats were deeply anesthetized with sodium pentobarbital (60 mg/kg), and the L4-L6 spinal cord was quickly harvested and stored at −80°C before being subjected to the following procedure. Samples were homogenized in Tris buffer with a cocktail of proteinase inhibitors and phosphatase inhibitors and were subjected to SDS-PAGE, followed by electrophoretic transfer onto polyvinylidene fluoride (PVDF) membranes. The membranes were placed in the block buffer at room temperature for 1 h and incubated with one of the following primary antibodies: anti-HCN2 (Proteintech, Wuhan, China), anti-NR2B (Proteintech), anti-pCaMKII (Cell Signalling Technology, USA); anti-CaMKII (Cell Signalling Technology); anti-pCREB (Cell Signalling Technology); anti-CREB (Cell Signalling Technology) at 4°C for 12 h, and then incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Proteintech) for 1 h. Quantification of the band for each protein was performed with Image J software.

To identify temporal expression of Shh signaling (Shh, Ptch1, Smo, Gli1), whole-cell protein extract lysates were used. To identify the activation of Shh signaling, nuclear extracts were prepared using an NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce Biotechnology) according to the manufacturer’s instruction. L4-L5 spinal cord segments were quickly removed from deeply anesthetized rats and homogenized in ice-cold RIPA lysis buffer containing a cocktail of protease inhibitors. Total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to 0.2 µm polyvinylidene difluoride membrane. The following primary antibodies were used: anti-Shh (1:2000, Abcam), anti-Ptch1(1:1000, Sigma), anti-Smo (1:2000, Abcam), anti-Gli1 (1:2000, Abcam), anti-p-GluN2B (Tyr1472) (1:500, Millipore), anti-p-CaMKII (1:1000, Cell signaling Tech), anti-p-CREB (1:1000, Cell signaling Tech), anti-Histone H3 (1:1000, Abcam), and anti-GAPDH (1:10,000, Sigma). The filters were developed using ECL reagents (Perkin Elmer) with secondary antibodies from Millipore Bioscience Research Reagents. Data were analyzed with a Molecular Imager (Gel DocTM XR, 170–8170) and the associated software Quantity One-4.6.5 (Bio-Rad Laboratories).