Hippocampal Protein Isolation and Western Blot Analysis

Protein isolation from hippocampal tissues was performed as previously (Zhou et al., 2010 (link)) described for either total cell isolates or cytosolic subcellular fractionated samples. Protein fraction was collected and homogenized on ice in RIPA buffer with protein concentration assessed and normalized with a Bradford protein assay kit (Beyotime, Shanghai, China). The following primary antibodies were used: anti-CHOP (1:200, Abcam), anti-CREB (1:200, Abcam), anti-Bim (1:200, Abcam), and anti-Cytochrome C (1:300, Abcam), anti-cleaved-caspase-3 (1:500, Cell Signaling Technology). Primary antibody detection was performed using secondary antibodies conjugated to horseradish peroxidase (Jackson Immuno Research). GAPDH expression in the same membrane was simultaneously determined as an internal reference. Western blots were imaged with a luminescent analyzer (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and quantification performed using Image Quant TL (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).