Western blot

Equal amounts of protein were loaded on sodium dodecyl sulfate–polyacrylamide (SDS-PAGE) TGX Stain-Free FastCast gel electrophoresis (TGX Stain-Free FastCast Acrylamide Kit, 7.5%, 161-0181 and 12%, 161-0185, Bio-Rad). According to BioRad protocol, after electrophoresis (30 min, 300 V), gels containing proteins were activated to UV light with Chemidoc imaging systems (12003153, Bio-Rad) using “Stain free gel activation (45 s)” program. Then, gels were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes at 1.3 A constant, up to 25 V using Turbo Transblot technology (1704150, Biorad). After blocking non-specific binding sites for 45 min, with a 5% solution of non-fat dry milk or 7.5% BSA in Tris Buffered Saline TBS (20 mM Tris/HCl, 137 mM NaCl, pH 7.4) containing 0.1% Tween 20 (TBST), membranes were probed with an anti-BDNF (1/3000, rabbit monoclonal, ab108319, Abcam), anti-vWF (1/2000, rabbit monoclonal, F3520, Sigma-Aldrich), anti-MyoD-1 (1/500, mouse monoclonal, ab16148, Abcam), anti-PAX 7 (1/2000, mouse monoclonal, ab218472, Abcam), anti-SYN (1/3000, rabbit polyclonal, RB-1461-P1, Interchim), anti-p-TrkB^Tyr816 (1/1000, rabbit polyclonal, ABN1381, Millipore), anti-p-eNOS^Ser1177 (1/1000, mouse monoclonal, 612,383, BD Biosciences) antibody for 1h30 at room temperature. Membranes were then incubated at room temperature (1 h) with horseradish peroxidase antibody [111-035-144 (anti-rabbit) or 115-035-166 (anti-mouse), 1/10000 to 1/50000 according to the protein, Jackson ImmunoResearch]. After antibody incubation, membranes were placed in Chemidoc imaging systems and a stain free image of the blot was captured to control the total protein loading and normalize data. Protein-antibody complexes were visualized using the enhanced chemiluminescence western blotting detection system (ECL 2, 1151-7371, Fisher Scientific) and Chemidoc imaging systems. Band densities were finally analyzed with ImageLab software (Bio-Rad) and standardized on total protein. The appropriate amounts of total proteins to be analyzed were previously determined from concentration (increasing amounts of proteins)/response (optical density of the band) curves from two rats both belonging to a particular group (on the same gel). All gels were run in triplicate.