Anti shc1

Western blot analysis was performed as previously described [22] (link). After sample preparation, equal amounts of a sample protein (50 μg) were loaded onto an SDS-PAGE gel. After electrophoresis, the samples were transferred onto a nitrocellulose membrane. Then, the membrane was blocked for 2 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: anti-apoE (1:500, Abcam, Cambridge, MA, USA), anti-Iba-1 (1:1000, Wako, USA), anti-APP (1:1500, Abcam, Cambridge, MA, USA), anti-myelin basic protein (MBP, 1:1000, Abcam, Cambridge, MA, USA), anti-LRP1 (1:1000, Abcam, Cambridge, MA, USA), anti-Shc1 (1:2000, Abcam, Cambridge, MA, USA), anti-PI3K (1:1000, Cell signaling, USA), anti-Akt (1:1000, Cell signaling, Danvers, MA, USA), anti-phospho-Akt (p-Akt, 1:1000, Cell signaling, Danvers, MA, USA), anti-CD16 (1:1500, Santa Cruz, Dallas, TX, USA), anti-iNOS (1:500, Abcam, Cambridge, MA, USA), anti-CD206 (1:1500, Santa Cruz, Dallas, TX, USA), and anti-β-actin (1:5000, Santa Cruz, Dallas, TX, USA). Appropriate secondary antibodies (1:5000, Santa Cruz, Dallas, TX, USA) were selected to incubate with the membrane for 2 h at room temperature. Then, blot bands were visualized with an ECL reagent (Amersham Biosciences UK Ltd., PA, USA). Non-saturated bands were selected to perform densitometry quantification using Image J software (Image J 1.51, NIH, USA).