Western Blot Analysis of Neural Proteins

Western blotting was performed as previously described [6 (link)]. Briefly, after brain protein sample preparation using RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA), equal amounts of protein were loaded on an SDS-PAGE gel and run using electrophoresis and then transferred to a nitrocellulose membrane. The membrane was blocked and incubated overnight at 4 °C with the following primary antibodies: goat anti-TREM2 (1:1000, Abcam, Cambridge, MA, USA), rabbit anti-PI3K (1:1000, Cell signaling, Danvers, MA, USA), rabbit anti-phosphorylated Akt (p-Akt, 1:1000, Cell signaling), rabbit anti-Akt (1:1000, Cell signaling), rabbit anti-TNF-α (1:1000, Abcam), rabbit anti-IL-1β (1:1000, Abcam), anti-Bcl-2 (1:2000, Abcam), anti-Bax (1:4000, Abcam), and goat anti-β-actin (1:5000, Santa Cruz Biotechnology). Appropriate secondary antibodies (1:3000, Santa Cruz; 1:5000, Abcam) were selected to incubate with the membrane for 2 h at room temperature. The bands were probed with an ECL Plus chemiluminescence regent Kit (Amersham Biosciences, Arlington Heights, PA, USA) and visualized with the image system (Versa Doc, model 4000, Bio-Rad, Hercules, CA, USA). Relative density of the protein immunoblot images were analyzed by ImageJ software (ImageJ 1.4, NIH, Bethesda, MD, USA).