Western blot analysis

Protein levels of GFAP, EAAT1, EAAT1, and NeuN were measured by Western blotting. The protocols were performed as previously described [17, 18]. Briefly, proteins were extracted from PFC tissue using a tissue protein extraction kit (CWBIO, #CW0891), and total protein concentrations were measured by using a BCA protein concentration determination kit (Thermo, #23227). Proteins were separated on 10% SDS-PAGE gels and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, #IPVH00010). After blocking with 5% skim milk solution for 2 h at room temperature, the membranes were incubated with primary antibodies at 4 °C overnight (GFAP, Abcam, #ab7260, 1:500 dilution; EAAT1, Cell Signaling, #5684, 1:1000 dilution; EAAT2, Cell Signaling, #3838, 1:1000 dilution; NeuN, Abcam, #ab177487, 1:500 dilution). After washing with TBST three times for 5 min each time, membranes were incubated with appropriate HRP-conjugated secondary antibodies. The protein bands were obtained using enhanced chemiluminescence (ECL), and the optical density of the protein bands was measured using GIS1D 4.2 image analysis software (#Tanon-5200, Tanon). The intensity of the protein bands was normalized to β-actin.