Anti bdnf

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.

Mouse brains were dissected on ice and then homogenized in a solubilization buffer containing 20 mM HEPES (pH:7.4), 1 mM EDTA, 1 mM PMSF, 250 mM sucrose, and protease inhibitor cocktail using a polytron at the lowest speed. The homogenates were centrifuged 20 min at 100,000 × g at 4°C. Supernatants were resupended in 20 mM HEPES (pH: 7.4), 1 mM EDTA and stored at −20°C until further use. Solubilized proteins were loaded at 50 μg in SDS buffer, separated on 10% SDS PAGE gels and then transferred to a nitrocellulose membrane.
Membranes were incubated with rabbit polyclonal Anti-BDNF (1:1000) (GeneTex, United States), Anti-Phospho(Tyr705)TrkB (1:500), Anti-TrkB (1:1000) (Signalway Antibody, United States), Anti-K2P2.1 (TREK-1) (1:500) and anti-ProBDNF (1:400) (Alomone Labs, Israel), Anti-Furin (1:1000) (SantaCruz Technologies, United States), Anti-Phospho(Ser133) CREB (1:500) and mouse monoclonal Anti-CREB (1:1000) (CST, United States), mouse monoclonal Anti-beta-Actin (1:5000) (SantaCruz Technologies, United States) over night at 4°C. Afterwards, membranes were incubed 30 min with secondary antibody (related to species of first antibody) coupled HRP. Protein bands were revealed, images were acquired with FX Fusion (Vilber) and analyzed with ImageJ (US NIH, Bethesda, MD, United States) (Schneider et al., 2012 (link)).