Brain tissues were collected and stored at −80 °C after being perfused with cold PBS (0.1 M, pH 7.4) at 12 h, 1 day, 3 days, 5 days, 7 days for the time course study, and at 3 days for the mechanistic study after GMH induction, respectively. Western blot was performed as previously described [25 (link)]. After sample preparation, 50 μg protein per sample was loaded onto 10–12% SDS-PAGE gels, ran for 90 min at 100 V, and was transferred onto 0.2 mm or 0.45 mm nitrocellulose membranes at 100 V for 120 min (Bio-Rad) [24 (link)]. The membranes were blocked for 2 h in 5% non-fat milk in Tris-buffered saline with 0.1% tween20, followed by overnight incubation at 4 °C with the following primary antibodies: anti-human NT-4 (ThermoFisher, USA), anti-NT-4 (Santa Cruz, USA), anti-TrkB (Abcam, USA), anti-pTrkB (ThermoFisher, USA), anti-PI3K(Abcam, USA), anti-Akt (Abcam, USA), anti-pAkt (Abcam, USA), anti-FoxO1(CST, USA), and anti-IL6 (Santa Cruz, USA). The same membranes were probed with actin (Santa Cruz, USA) as internal loading controls. Appropriate secondary antibodies (Santa Cruz, USA) were incubated with membranes for 2 h at room temperature. Bands were visualized using ECL Plus Chemiluminescence kit (Amersham Biosciences, USA) and quantified through ImageJ 4.0 (Media Cybernetics). And we have the gel scans of all proteins in this study (Supplementary Fig. 3 ).
Free full text: Click here
