P akt

Primary antibodies targeting the following proteins were used for western blot and tissue sections: FGF9 (Abcam, EPR19937), Olig1 (Millipore, MAB5540), NeuN (Millipore, ABN78), Iba-1 (Abcam, ab5076), anti-GFAP (Millipore, MAB360), anti-Calbindin (Sigma, C2724), Caspase-3 (Santa Cruz, sc-373730), GABA (Sigma, A2052), VGAT (SYSY, 131011), Calbindin (Sigma, C2724), GAPDH (Abcam,ab128915), Adcy5/6 (Santa Cruz, sc-514785), p-ERK (Cell Signaling, #4370), ERK (Cell Signaling, #4695), p-Akt (Immunoway, YP006), Akt (Immunoway, YT0176), and β-actin (Proteintech, 60008-1-Ig). Secondary antibodies for western blot were from Rockland Immunochemicals, USA, and fluorescein isothiocyanate secondary antibodies were from Jackson ImmunoResearch, West Grove, PA. Hoechst stain (1:100, Invitrogen), RNeasy Lipid Tissue Mini kit (QIAGEN, 74804), SQ22536, bupivacaine-HCl, pentylenetetrazol, GABA, and Glu were purchased from Sigma (Madrid, Spain).

Total tissue lysates were separated on SDS-PAGE and electrotransferred onto polyvinylidene fluoride membranes (0.22 μm) by wet blotting (Bio-Rad). Primary antibodies detected the following proteins: β-actin (1:100,000, Cat No. AC026, Abclonal), α-tubulin (1:50,000, Cat No. 66031-1-Ig, Proteintech), CX3CL1 (1:1000, Cat No. A14198, Abclonal), GNB5 (1:1000, Cat No. A4447, Abclonal), AKT2 (1:5000, Cat No. ab131168, Abcam), p-AKT (1:1000, Cat No. YP0006, Immunoway), NF-κB (1:2000, Cat No. 66535-1-Ig, Proteintech), p-NF-κB (1:1000, Cat No. 93H1, Cell Signaling Technology), Bad (1:1000, Cat No. ab32445, Abcam), Bcl-2 (1:2000, Cat No. ab182858, Abcam), occludin (1:3500, Cat No. 13409-1-AP, Proteintech), claudin-1 (1:4500, Cat No. 13050-1-AP, Proteintech), which were then incubated with goat anti-rabbit IgG and goat anti-mouse IgG and HRP-conjugated(1:5000, Cat No. CW0103S, CW0102S, CWBIO). Then, stripping buffer (CW0056M, CWBIO) was used for the replacement of antibodies. The density of each protein was detected by an ECL detection kit (Solarbio).