Western Blot Analysis of Brain Proteins

Western blotting was performed on fetal brain tissue and offspring hippocampus according to the method as previously described by our research group (Wu ZY et al., 2018 (link); Li XY et al., 2017 (link)). The whole brains of fetuses were removed immediately when the female rats were under cesarean section. Hippocampus of offspring rats was harvested at P33 after the behavior tests. Fetal brain tissue and hippocampus homogenates (n = 3 per group) were prepared for determination of CB1R, CDK5, p35, p25, tau, p-tau, and GAPDH levels. Protein levels were examined by incubating hippocampus homogenates with antibodies to CB1R (1:1000, Cell Signaling Technology, 93,815, United States), CDK5 (1:1000, Cell Signaling Technology, 14,145, United States), p35 (1:1000, Cell Signaling Technology, 2,680, United States), p25 (1:1000, Cell Signaling Technology, 2,680, United States), tau (1:1000, Cell Signaling Technology, 46,687, United States), p-tau (1:1000, Cell Signaling Technology, 20,194, United States), and GAPDH (1:1000, Cell Signaling Technology, 2,118, United States). If the target protein is phosphorylation index (tau,p-tau), phosphatase inhibitors should be added in addition to PMSF. Visualization of the bands was performed with enhanced chemiluminescence and quantification was carried out with ImageJ (NIH Image, United States).