Western blotting

Western blotting was performed to test the expressions of p11, BDNF, proBDNF, TrkB and p-TrkB in the hippocampus of rat or cultured neurons. After the behavioral tests, the rats were anesthetized with sodium pentobarbital (60 mg kg⁻¹), and the hippocampus was removed and homogenized on ice. The hippocampal neural cells were dissociated by cell lysis buffer. The BCA assay was used to determine the concentration of proteins. All the normalized samples were separated by 12 or 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes, blocked by 3% bovine serum albumin and incubated with primary antibodies overnight at 4 °C, including p11 (1:500, Abcam, Cambridge, UK), BDNF (1:1000, Abcam), proBDNF (1:800, Abcam), TrkB (1:500, Abcam), p-TrkB (1:500, Abcam) and tubulin (1:1000, Abcam). The polyvinylidene fluoride membranes were washed with Tris-buffered saline plus Tween 20 and then incubated with second antibodies for 1 h at room temperature (rabbit or mouse anti-goat 1:8000). ImageJ software was used to calculate the gray value of immune reactivity.