Rabbit anti pi3k

Neuronal progenitors from wild-type and twitcher mice were grown under differentiation conditions for 7 DIV, and cultured in the presence or absence of IGF-1 (100 ng/ml) for 24 h. Cells were fixed in 4% paraformaldehyde/PBS and then blocked in 5% BSA/PBS. Cells were incubated with 1:100 dilution of cholera-toxin–Alexa-Fluor-488 (Molecular Probes) and 1:100 dilution of rabbit anti-PI3K (Cell Signaling) at 4°C overnight. After incubation with anti-rabbit IgG-Alexa-Fluor-546 (Molecular Probes; 1:1500 dilution in blocking buffer), samples were confocally imaged in an upright Leica TCS DM5500Q confocal microscope (Leica Biosystems, Buffalo Grove, IL, 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).

The total protein from the indicated cells was quantified and solubilized in Laemmli buffer. Target proteins were probed on PVDF membranes with the corresponding primary antibodies: rabbit anti‐PDK1 (Abcam, Cambridge, UK, ab52893), rabbit anti‐P‐AKT (Cell Signaling Technology, Denver, CO, USA, #4060), rabbit anti‐PI3K (Cell Signaling Technology, #4249), mouse anti‐Caspase‐3 (Abcam, ab13585), and mouse anti‐beta Actin (Abcam, ab 8226) in TBST containing 5% BSA at 4 °C overnight. The membrane was then washed with TBST, followed by adding the corresponding secondary antibody. The membrane was finally incubated with ECL solution and developed using a Roche exposure system. The quantification and comparison of grayscale density of protein bands was carried out using ImageJ software (Shanghai KangChen Bio‐tech Inc.).

According to the manufacturer’s instructions, the protein extracts of nucleus and cytoplasm (three mice in each group) were prepared using nuclear and cytoplasmic protein extraction kit (Beyotime Institute of Biotechnology, Shanghai, China). The total protein was assayed using BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Proteins (50 μg) were subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After blocked with 5% nonfat milk for 1 h, the membranes were incubated overnight at 4 °C with the primary antibodies for cytosolic fractions: rabbit anti-TH antibody (1:1000, Millipore Corporation), rabbit anti-phospho-PI3K (1:1000, Cell Signaling Technology), rabbit anti-PI3K (1:1000, Cell Signaling), rabbit anti-phospho-AKT (1:1000, Cell Signaling), rabbit anti-AKT (1:1000; Cell Signaling), anti-GCLC (1:1000, Abcam), anti-GCLM (1:2000, Abcam), anti-Heme Oxygenase 1 (1:2000, Abcam), anti-Keap1 (1 μg/ml, Abcam), rabbit anti-β-Actin (1:1000; Cell Signaling), and the primary antibodies for nuclear fractions: anti-Lamin B1 (0.1 μg/ml, Abcam), anti-Nrf2 (1:1000, Abcam). The membranes were incubated with the horseradish peroxidase-labeled secondary antibody (1:2000). Bands were investigated using the ECL detection reagents (Beyotime Institute of Biotechnology, Shanghai, China).

The muscle tissues were homogenized with lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl, 1 mM ethylene glycol tetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₂VO₄ and 100 mM NaF), and then centrifuged at 12,000 rpm for 10 minutes. The protein was separated on sodium dodecyl sulfate-polyacryl-amide gel and transferred to the nitrocellulose membrane. GAPDH antibody (1:3,000 Cell Signaling Technology, Beverly, MA, USA), rabbit anti-myostatin antibody (1:1,000; Millipore, Billerica, MA, USA), rabbit anti-PI3K (1:1,000; Cell Signaling, USA) and rabbit anti-AKT (1:1,000; Cell Signaling, USA) were used as primary antibodies. Band detection was performed using the enhanced chemiluminescence detection kit (Santa Cruz Biotechnology, Dallas, CA, USA).