Western Blot Analysis of Hippocampal Proteins

Western immunoblotting was carried out as previously described (Silasi et al., 2004 (link); Kovalchuk et al., 2016a (link),b (link),c (link)). In brief, hippocampal tissues were sonicated in ice-cold 1% SDS and immediately boiled. Protein concentrations were determined using the Bradford assay (BioRad, Hercules, CA). Equal amounts of protein (10–30 μg) were separated by SDS-PAGE into slab gels of 10–15% polyacrylamide and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Baie d'Urfé, Quebec). Eight membranes were prepared. The membranes were incubated with primary antibodies against 4-HNE, AKT 1, NPAS4 (1:1,000, Abcam), ERK1/2, FOSB, PCNA (1:1,000, Cell Signaling), and actin (1:2,000, Abcam) overnight at 4°C. Primary antibody binding was detected using horseradish peroxidase-conjugated secondary antibodies and the Enhanced Chemiluminescence Plus System (Amersham Biosciences, Baie d'Urfé, Quebec). Chemiluminescence was detected using a FluorChem HD2 camera with FluorChem software (Cell Biosciences). The membranes were stained with Coomassie blue (BioRad, Hercules, CA) to confirm equal protein loading. Signals were quantified using NIH Image J64 software and normalized relative to actin or Coomassie staining.