Western Blot Validation of DPYSL2 and BDNF Expression

Western blot (WB) was performed in order to validate peculiar increase of expression of both DPYSL2 and BDNF found in hill with respect to plain and Control samples with 2-DE and RT-PCR, respectively. Aliquots of protein samples (5 μg for DPYSL2 and 50 μg for BDNF) were mixed with Laemmli solution, run in 4–15% polyacrylamide gels (Mini-PROTEAN^® Precast Gels, Biorad, Hercules, CA, USA) using a mini-Protean Tetracell (Biorad, Hercules, CA, USA) and transferred onto nitrocellulose membranes (0.2 μm) using a Trans-Blot Turbo transfer system (Biorad) as previously described [45 (link)]. Anti-DPYSL2 (Cell Signaling Technology, Beverly, MA, USA) and anti-BDNF (Genetex, Irvine, CA, USA) antibodies were used at 1:1000 dilution. Moreover, an anti-β-actin (Merck KGaA, Darmstadt, Germany), was used for internal normalization. HRP-goat anti-rabbit secondary antibody was used at 1:10,000 dilution. Immunoblots were developed using the enhanced chemiluminescence detection system (ECL). The chemiluminescent images were acquired using LAS4010 (GE Health Care Europe, Upsala, Sweden). The immunoreactive specific bands were quantified using Image Quant-L software.