Anti crmp2

The following antibodies were used: anti-GAPDH (1:5000) and anti-CDKL5 (1:500) (Sigma); anti-phosphorylated Erk1/2 (1:1000), anti Erk1/2 (1:1000), anti-phospho-AKT-Ser⁴⁷³ (1:1000), anti-phospho-AKT-Thr³⁰⁸ (1:1000), anti-AKT (1:1000), anti-phospho-GSK-3β-Ser⁹ (1:1000), anti-GSK-3β (1:1000), anti-phospho-CRMP2-Thr⁵¹⁴ (1:1000) and anti-CRMP2 (1:1000) (Cell Signaling Technology); anti β-catenin (1:1000; BD Transduction Laboratories); anti-phospho-CREB-Ser¹³³ (1:1000) and anti-CREB (1:1000) (Upstate Biotechnology). For the preparation of total cell extracts, cells were lysed in RIPA buffer (Tris–HCl 50 mM, NaCl 150 mM, Triton X-100 1%, sodium deoxycholate 0.5%, SDS 0.1%, protease and phosphatase inhibitors cocktails 1%; Sigma). For the preparation of tissue extracts, the hippocampi from P19 mice were homogenized in RIPA buffer. Extracts were immediately processed by Western blot or kept frozen (− 80 °C) until assayed. Sample protein concentration was estimated using the Lowry method (Lowry et al., 1951 (link)). Equivalent amounts (50 μg) of protein were subjected to electrophoresis on a 10% SDS-polyacrylamide gel. Densitometric analysis of digitized images was performed using Scion Image software (Scion Corporation, Frederick, MD, USA) and intensity for each band was normalized to the intensity of the respective total protein levels or GAPDH band.

Soluble Nogo-66 protein was obtained via SUMO fusion in Escherichia coli from our laboratory (16 (link)). Y-27632 and the NEP1-40 were obtained from Merck KGaA and Tocris Bioscience, respectively. The Cy3-conjugated anti-rabbit secondary antibody was obtained from Abcam (cat. no. ab6939). Rabbit monoclonal IgG anti-APP was purchased from LifeSpan BioSciences, Inc. In addition, the following primary antibodies were obtained: Anti-β-secretase 1 (BACE1; cat. no. 5606; Cell Signaling Technology, Inc.), anti-ROCK2 (cat. no. ab125025; Abcam), anti-phosphorylated (p)-collapsin response mediator protein-2 (CRMP2; Thr514; cat. no. 9397; Cell Signaling Technology, Inc.), anti-CRMP2 (cat. no. 35672; Cell Signaling Technology, Inc.), anti-GAPDH (cat. no. BS72410; Bioworld Technology, Inc.) and anti-microtubule-associated protein 2 (MAP2; cat. no. 05-346; Merck KGaA). HRP-conjugated goat anti-rabbit IgG antibody (cat. no. E030120-01; Earthox Life Sciences) was used. All reagents and drugs used were of analytical grade.