Anti ptch1

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).

Proteins were extracted from the ipsilateral T4-5 spinal cord dorsal horn (SDH), and the supernatant protein concentrations were detected with the BCA protein assay kit (Abcam). The equal number of sample proteins was separated on sodium dodecyl sulphate–polyacrylamide gels. Subsequently, they were transferred onto the polyvinylidene fluoride membrane, which was blocked with 5% non-fat milk and then incubated with primary antibodies overnight at 4°C. The primary antibodies included anti-Shh (1:2000, GeneTex, San Antonio, USA), anti-Ptch1 (1:1000, Sigma, St. Louis, USA), anti-Smo (1:2000, Abcam), anti-Gli1 (1:2000, Abcam), anti-BDNF (1:1000, GeneTex), anti-p-TrkB (1:1000, GeneTex), anti-TrkB (1:2000, GeneTex), anti-p-PI3K (1:1000, GeneTex), anti-p-Akt (1:1000, GeneTex), anti-Histone H3 (1:2000, GeneTex), and anti-GAPDH (1:10,000, GeneTex). Finally, the membranes were rinsed and the proteins were detected with the enhanced chemiluminescence method.