Immobilon western hrp detection substrate

Western blotting analysis was performed as described previously (Ohnishi et al., 2011 (link)). Cell pellets or hippocampal tissues were lysed in 2 x SDS sample buffer (125 mM Tris-HCl pH 6.8, 4% SDS, 10% glycerol) and sonicated. After centrifugation (15,000 x g, 4°C, 15 min), protein concentrations of the supernatants were determined using the DC Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Samples were diluted in 1 x SDS sample buffer with 2-mercaptoethanol and bromophenol blue (BPB), boiled, separated on 12.5% SDS-polyacrylamide gels, and then transferred to Immobilon-P membranes (Merck Millipore). The membranes were washed with TBST buffer [10 mM Tris (pH 7.5), 150 mM NaCl and 0.1% Tween 20], and blocked in TBST buffer containing 5% BSA. Membranes were treated with rabbit monoclonal anti-FOSB (5G4) (1:1000; Cell Signaling Technology Japan, K.K., Tokyo, Japan) or mouse monoclonal anti-GAPDH (1:100000; Merck Millipore), and then horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:10000; Merck Millipore) or HRP-conjugated anti-mouse Ig (1:2500; BD Biosciences, San Jose, CA, USA). Membranes were washed in TBST buffer and bound antibodies were visualized using Immobilon® Western HRP Detection Substrate (Merck Millipore). Digitized images were obtained with the LAS1000 Plus Luminescent Image Analyzer (Fuji Film, Tokyo, Japan).