Rabbit anti rat β actin

The total cellular protein was extracted from hippocampal tissue or treated cells using a protein extraction kit, and the concentrations were measured using a Bicinchoninic Acid Kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein were separated by 10%–12% SDS-PAGE (Bio-Rad) and transferred onto polyvinylidene fluoride (PDVF) membranes (Millipore, Billerica, MA, USA). After subsequently blocking in 5% nonfat dry milk for 2 h, the membranes were incubated overnight at 4°C with the relevant primary antibody, followed by incubation with secondary antibodies for 1.5 h at 37°C. The primary rabbit anti-rat monoclonal antibodies against CaM, CaMKK, p-CaMKIV, and p-tau were all obtained from Abcam (1 : 1000, Cambridge, UK). The secondary antibody, HRP-conjugated goat anti-rabbit IgG, was obtained from AmyJet Scientific Inc. (1 : 20,000, Abbkine, Wuhan, China). Rabbit anti-rat β-actin (1 : 2000, Abbkine, AmyJet Scientific Inc.) was used as an internal control. The protein bands were visualized using electrogenerated chemiluminescence (ECL; Thermo Fisher Scientific) and analyzed on a gel imager (FluorChem M, ProteinSimple, USA).

The total protein was extracted from hippocampus tissue using a radio-immunoprecipitation assay (RIPA) buffer containing protease inhibitors (Abmole biotechnology, Shanghai, China). The 20 μg extracted proteins were separated by 10% SDS-PAGE (Bio-Rad) and transferred onto polyvinylidene fluoride (PDVF) membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% nonfat dry milk for 2 h and incubated overnight at 4°C with the relevant primary antibodies. The primary antibodies were anti-BDNF(1 : 1000, novus biologicals, Beijing, China), anti-caspase-3 (1 : 1000, Affinity, Liyang, China), anti-Bax (1 : 1000, Abcam, Cambridge, UK), anti-Bcl-2 (1 : 1000, Abcam, Cambridge, UK), anti-p-CREB (1 : 1000, Cell Signaling Technology, MA, USA), anti-TrkB(1 : 1000, Cell Signaling Technology, MA, USA), and anti-p-Akt (1 : 2000, Cell Signaling Technology, MA, USA). The membrane was incubated with the horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) (1 : 20,000, Abbkine, Wuhan, China). The membrane was visualized by the proteins by ECL reagent and analyzed by ImageJ software. Rabbit anti-rat β-actin (1 : 2000, Abbkine, Wuhan, China) was used as an internal control.