Western blot analysis
The protein concentrations of lysates from cells and mice brains were
detected by the Bradford method and then separated on 8-15% SDS-PAGE.
After transferring the gels to polyvinylidene difluoride (PVDF,
Millipore Corp., Billerica, MA, USA) membranes, the membranes were
incubated with 5% skim milk or 3% BSA. Membranes were incubated with
the primary antibody (1:1000) overnight, and there were followed by
horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000)
for 1 h. Immunoreactive bands were detected with SuperSignal West Femto
Maximum Sensitivity Substrate kit (Thermo Scientific, Pierce
Biotechnology, Rockford, Illinois, USA) using the LAS-4000 mini system
(Fujifilm, Japan). The intensities of the bands were normalized to the
GAPDH intensity using Multi Gauge software (Fujifilm). All experimental
procedures conformed with British Journal of Pharmacologyguideline (Alexander et al., 2018).