Hippocampus Protein Extraction and Western Blot

Western blot was performed, as described by Ma [75 (link)]. Total protein was extracted from the whole hippocampus or primary cultures using RIPA buffer (Solorbio Life Sciences, Beijing, China, # R0010). Samples were homogenized using a Pro Homogenizer, and the protein concentration was determined using the bicinchoninic acid assay (BCA) with bovine serum albumin as a standard. Briefly, 30 μg of proteins was loaded per lane on an 8–10% gradient acrylamide gel, and the proteins were transferred to Immobilon-p transfer membranes (Millipore) and incubated with the following primary antibodies: KL (1:1000, ab203576, Abcam) and GAPDH (1:10000, ZSGB-BIO). After incubation with the corresponding secondary antibodies, membranes were visualized using an luminescent imaging system (Tanon, China). The signal for each target protein was normalized to the GAPDH signal before being analyzed.