Rabbit anti phosphorylated akt p akt

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).

At each post-ICH time point, the brain samples were collected and prepared for western blot analysis as previously described [8 ]. Briefly, the ipsilateral/right brain hemisphere sample was prepared using RIPA lysis buffer (sc-24948, Santa Cruz Biotechnology). Then, 4 μL of protein sample was loaded onto an SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk and incubated with the following primary antibodies: rabbit anti-CCR4 (1:1000, GTX53474, Gene Tex, USA); rabbit anti-CCL17 (1:1000, ab182793, Abcam); rabbit anti-PI3K (1:1000, Cell Signaling, Danvers, MA, USA); rabbit anti-AKT (1:1000, Cell Signaling); rabbit anti-phosphorylated AKT (p-AKT, 1:1000, Cell Signaling); rabbit anti-Foxo1 (1:1000, ab179450, Abcam); goat anti-Iba-1 (1:1000, ab5076, Abcam); mouse anti-MPO (1:1000, sc-390109, Santa Cruz Biotechnology); rabbit anti-IL-1β (1:1000, ab9722, Abcam); rabbit anti-TNF-α (1:1000, ab6671, Abcam); anti-Bcl-2 (1:2000, Abcam); anti-Bax (1:4000, Abcam); and mouse anti-β-actin (1:3000, sc-47778, Santa Cruz Biotechnology) overnight at 4 °C. Membranes were incubated with corresponding secondary antibody (1:3000, Santa Cruz; 1:5000, Abcam) for 2 h at room temperature. The relative density of the protein bands was quantified by the ImageJ software (ImageJ 1.4, NIH, Bethesda, MD, USA).