Anti probdnf

The ELISA kit to measure corticosterone was from Enzo Life Science. Rabbit polyclonal antibodies anti-TREK-1 and anti-proBDNF were from Alomone Labs (Israel). Rabbit polyclonal antibodies anti-furin and anti-NaKATPase, the mouse monoclonal anti-beta-actin and goat anti-TGN38 and anti-DCX antibodies were from SantaCruz Technologies (United States). Rabbit polyclonal antibodies anti-TrkB and anti-phospho-(Tyr 705)-TrkB were from Signalway Antibody (United States). Rabbit polyclonal antibodies against BDNF and plasminogen were from GeneTex (United States). Mouse monoclonal anti-BrdU antibody was from Becton Dickinson (United States). Anti-rabbit, anti-mouse, and anti-goat secondary antibodies were from Cell Signaling Technologies. HRP substrate (Immobilon Forte, Millipore), Nitrocellulose (BioTrace NT, Life Sciences), SDS-PAGE gels and Mini Protean apparatus were from Biorad. BrdU was from Interchim (France). Neurobasal medium, B-27 and Penicillin-Streptomycin were from Thermo Fisher Scientific. Poly-D-Lysine was from Sigma-Aldrich. Patch clamp was performed on a pClamp 8.2 apparatus (Molecular Devices)

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)).