Goat anti rabbit igg

Brain samples were obtained 4 weeks after stroke and were immediately dissected into the right (R; ischemic) and left (L; non-ischemic) hemispheres. Tissue was homogenized with Dounce homogenizers in cold triton lysis as previously described27 (link). A total 30μg of protein was loaded in each well and resolved on 4%–15% SDS gels and transferred to a PVDF (polyvinylidene difluoride) membrane. Total myelin, BDNF, pTrkb, Trkb synapsin, p-synapsin, Akt, p-Akt P-MAPK (ERK 1/2), MAPK and actin were detected by the following antibodies; Myelin Basic Protein (1:1000 Abcam); BDNF (1:1000 EMD millipore ); pTrkB 1:500 (706, Santa Cruz), TrkB 1:1000 (Santa Cruz); Synapsin and p-synapsin (1:1000, CST); Akt and p-Akt (1:1000, CST); ERK1/2 and p-ERK1/2 (1:2500 CST) and actin (1:5000, Sigma) respectively. All blots were blocked with 5% non-fat dry milk or 4% BSA for 1h at room temperature and incubated overnight with primary antibodies at 4 °C. Secondary antibodies were either goat anti-rabbit IgG 1:10,000 (GE Healthcare Life sciences) or goat anti-mouse IgG 1:10,000 (GE Healthcare Life sciences). Thermo Scientific Super Signal West Pico Chemiluminescent Substrate was used for signal detection. Densitometry (n = 5-6 per group) was performed with Image J software.

Cells were lysed in Nonidet P-40 (NP40) lysis buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, and 1% NP40). Equal amounts of total protein, as determined by the BCA protein assay kit (Pierce, Rockford, IL, USA), were separated by SDS-PAGE on 8% polyacrylamide gels and transferred to Hybond ECL nitrocellulose membranes (GE Healthcare, Europe GmbH, Freiburg, Germany). Blots were stained with Ponceau S to ensure protein amounts were equal. For immunodetection, blots were soaked in 1% blocking reagent (Roche, Basel, Switzerland) in 0.05% Tween 20-PBS for 1 h and incubated with primary antibody in blocking buffer overnight at 4 °C. Blots were then washed in 0.05% Tween 20-PBS and incubated with either goat anti-mouse IgG (1:20,000; GE Healthcare) or goat anti-rabbit IgG (1:20,000; GE Healthcare) peroxidase-labeled antibodies in blocking buffer for 1 h. An enhanced chemiluminescent ECL system (GE Healthcare) was applied according to the manufacturer’s protocol. The experiments were performed in triplicate. Scanning densitometry of blots was analyzed using ImageJ software (Rasband, W.S., US National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij/).

EGFR-mt and EGFR-wt LA cells were treated with DMSO (Control) or AZD9291 (1 µM) for 24 h, then the nuclear proteins were extracted using Nuclear Extraction Kit (Active Motif, 40010) in biological triplicates. The protein concentrations were measured using DC Protein Assay Kit (Promega, 5000112JA). The methods of SDS-PAE, and immunoblotting were described previously^{16 (link)}. The primary (anti-histone H3ac (pan-acetyl) Ab (Active Motif, 39040), anti-Lamin A/C Ab (Active Motif, 39287)) and secondary (goat anti-rabbit IgG (GE Healthcare, NA931)) antibodies were used at a dilution of 1:1000. Immunoreactivity was detected using Fusion SoloS (M&S Instruments Inc., Japan).

Cells (5 × 10⁶ to 10⁷) were solubilized in RIPA lysis buffer containing 1% Nonidet P-40/0.1% sodium dodecyl sulfate (SDS) as described^{8 (link)}. For immunoprecipitation, cell lysates were incubated with 2 μg/ml of IV.3 anti-FcγRIIA, A77 anti-FcαRI or ZL7-4 anti-CD79a mAbs and immunoprecipitated overnight at 4 °C with Protein G-Sepharose (GE Healthcare). Samples were resolved by SDS polyacrylamide gel electrophoresis (10%), transferred to nitrocellulose membranes and immunoblotted with rabbit antibodies followed by goat anti-rabbit IgG (GE Healthcare) coupled to horseradish peroxidase. Membranes were developed by enhanced chemical luminescence treatment (Amersham Biosciences). All uncut Western blots are available in Supplementary Fig. 10.

NHP-derived BEC were lysed using ice-cold radio-immunoprecipitation assay (RIPA) or cell lysis buffer containing protease inhibitor cocktail (ThermoFisher) centrifuged at 15,000g for 15 min, and supernatant fractions were collected. Samples were added to SDS and loading buffer then denaturized by heat at 95 °C for 5 min. Denaturized samples were loaded into 4–12% Tris–Glycine SDS-page gels (Invitrogen), and left to migrate for 1 h at 180 V. Samples were then transferred onto polyvinylidene fluoride (PVDF) or nitrocellulose membranes using an iBlot 2 Dry Blotting System (Invitrogen) on the P0 program (20 V for 1 min, 23 V for 4 min, 25 V for 2 min). PVDF membranes were then rinsed with Tris-buffered saline with 0.1% Tween 20 (TBST) and blocked for 1 h in 5% non-fat dry milk in TBST (blocking buffer). Membranes were first probed overnight at 4 °C with primary antibodies in blocking buffer (anti-ITM2A polyclonal AF4876 and 18306-1-AP, EmGFP A11122, anti-HA 901509, anti α-tubulin T9026) and then probed with secondary antibodies diluted in TBST for 1 h at RT (1:10,000 diluted HRP-coupled goat anti-mouse IgG or goat anti-rabbit IgG, GE Healthcare). Following secondary antibody incubation, membranes were rinsed thoroughly with TBST, imaged using a LICOR Odyssey Imager and bands quantified using Multi-Gauge v3.0.