0.2 mm nitrocellulose membranes

The samples were homogenized and processed in a lysis buffer for Western analysis as previously described (Castrén et al., 2002 (link)). The protein concentration of the supernatant samples was determined using Biorad DC protein assay. The protein extracts (60 μg) were electrophoresed on 7.5% sodium dodecyl sulfate polyacrylamide minigels and transferred to 0.2 mm nitrocellulose membranes (Schleicher & Schuell) for 1 h at 400 mA. The membranes were washed 10 min in TBS, pH 7.4 (0.1 M Tris, 0.15 M NaCl) and blocked in 5% non-fat dry milk, in TBS with 0.1% Tween 20 (TBST) for 1.5 h. The incubation with rabbit anti-TrkB (1:1000, sc-11, Santa Cruz Biotechnology) at +4°C overnight was followed by washes in TBST and incubation with horseradish-peroxidase-conjugated secondary antibody (1:10000, Bio-Rad Laboratories) for 1.5 h at room temperature. Detection was performed using the enhanced chemiluminescence kit (ECL⁺⁺ kit, Amersham Biosciences) and Fuji LAS-3000 camera (Tamro Medlabs, Vantaa, Finland). Data were analyzed using NIH Image J software.