Signalboost immunoreaction enhancer kit

For Western blot analysis, cells were lysed in RIPA buffer supplemented with protease inhibitor cocktail (Complete, Roche). Hypoxia treated cells were lysed within the hypoxia chamber. Protein concentration was determined with the Pierce BCA protein assay kit (Thermo Scientific). Protein samples were boiled at 95 °C for 5 min, where after 50 μg was loaded per well in Nu-PAGE 4–12% Bis-Tris gels (Life Technologies) for SDS-PAGE, and then transferred to Protein nitrocellulose membranes (Schleicher and Schuell). Membranes were blocked for 1 h using Odyssey blocking buffer (LI-COR Biosciences), and incubated with the primary antibody, sometimes in combination with the SignalBoost Immunoreaction Enhancer Kit (EMD Millipore), overnight at +4 °C with end-over-end rotation. Immunoblots were visualized with the Odyssey Infrared Imaging system (LI-COR Biosciences) in accordance with the manufacturer’s recommendations. Antibodies are listed in the Supplementary information.

Cells were lysed in ice-cold TNTE buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 10% v/v glycerol, 5 mM EDTA) containing EDTA-free Complete Protease Inhibitor cocktail (Roche) and PhosSTOP (Roche). Lysates were cleared by centrifugation and resolved on NuPAGE Bis-Tris 4–12% gels (Life Technologies) followed by transfer onto a PVDF membrane (Millipore). After incubation with primary and secondary antibodies, the blots were developed by chemiluminescence using Immobilon Classico Western HRP substrate (Merck Millipore). Densitometry was performed with ImageJ software. For western blotting of weak signal antibodies, primary antibody was diluted with SignalBoost Immunoreaction Enhancer Kit (Merck Millipore, 407207) and blots were developed with Luminata Crescendo Western HRP substrate (Merck Millipore).

The CYP2D protein levels in the brain and liver microsomes of control and lurasidone-treated rats were estimated by Western immunoblot analysis, as described previously [15 (link)]. The total protein concentration in samples was determined in the microsomes using the Lowry methods [26 (link)]. All samples were heated in a Laemmli sample buffer for 5 min at 100 °C. Next, the microsomal proteins (10 μg) were separated using an SDS polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The blots were probed with the following primary antibodies: rabbit antirat CYP2D4 (1:1000) or rabbit antihuman CYP2D6 (1:2000). In order to dilute the primary and secondary antibodies, the specified solutions from the SignalBoost™ Immunoreaction Enhancer Kit (Merck Millipore, Burlington, MA, USA) were used. As a positive control, we used rat cDNA-expressed CYP2D4 (2.5 µg) or human CYP2D6 (1 µg). The blots were visualized using the Luminescent Image Analyzer LAS-1000 and Image Gauge 3.11 programs (Fuji Film, Tokyo, Japan). The results were normalized to β-actin protein level.

Protein extracts from cell lysates were obtained with RIPA buffer mixed protease inhibitor cocktail (Thermo scientific). Protein extractions were boiled in 4× Protein SDS-PAGE Loading Buffer (Takara) for 5 min and resolved on a SDS-PAGE, and then transferred onto 0.45-μm NC membrane (GE Whatman). Membranes with total FGFR proteins were blocked by 5% (w/v) non-fat dry milk (GE Healthcare), while those with phosphorylated FGFR1 protein were treated with 5% (w/v) bovine serum albumin (BSA) for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies including anti-FGFR1 polyclonal antibody (1:500 dilution, Cell Signaling Technology), anti-phosphorylated FGFR1 polyclonal antibody (1:1000 dilution, Lianke Biotechnology Ltd.), anti-FGFR4 monoclonal antibody (1:1000 dilution, Abcam; Epitomics), and anti-β-actin monoclonal antibody (1:5000 dilution, Sigma-Aldrich). After three washes with TBS containing 0.1% (v/v) Triton X-100 (TBST), they were incubated with goat anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 dilution, Cell Signaling Technology) for 1 h at room temperature. Final detection of protein was performed using the Signal Boost Immunoreaction Enhancer Kit (Merck Millipore). Protein levels were quantified using Quantity One software (Bio-Rad).

Ipsilateral dorsal lumbar (L4–L6) spinal cord and dorsal root ganglia (DRG) were collected immediately after decapitation on 2nd, 7th, and 14th days after CCI. Tissue was stored at −80°C until processing, which was described previously [28 (link)]. Blots were incubated overnight at 4°C with primary antibodies: anti-IBA-1 (rabbit anti-rat, 1 : 1000, Proteintech, Chicago IL, USA), anti-TLR2 (rabbit anti-rat, 1 : 2000, Novus Biological, Littleton CO, USA), anti-TLR4 (rabbit anti-rat, 1 : 1000, Proteintech, Chicago IL, USA), anti-MyD88 (rabbit anti-rat, 1 : 1000, Abcam, Cambridge, UK), and anti-TRIF (rabbit anti-rat, 1 : 500, Novus Biologicals, Littleton CO, USA) and for 1 h at RT with a corresponding secondary polyclonal HRP antibody (goat anti-rabbit IgG, 1 : 5000, Bio-Rad, Hercules, CA, USA). Both primary and secondary antibodies were diluted in solutions from SignalBoost Immunoreaction Enhancer Kit (Merck Millipore, Darmstadt, Germany). Immunocomplexes were detected using Clarity Western ECL Substrate (BioRad, Hercules, CA, USA) and visualized using a Fujifilm LAS-4000 fluoroimager system. The blots were stripped using Restore Western Blot Stripping Buffer (ThermoScientific, Waltham, MA, USA) for 15 minutes at RT and reprobed with an antibody against GAPDH (mouse anti-rabbit, 1 : 5000, Merck Millipore, Darmstadt, Germany) as a loading control.

Cells were lysed in ice-cold TNTE buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 10% v/v glycerol, 5 mM EDTA) containing EDTA-free Complete Protease Inhibitor cocktail (Roche) and PhosSTOP (Roche). Lysates were cleared by centrifugation and resolved on NuPAGE Bis-Tris 4–12% gels (Life Technologies) followed by transfer onto a PVDF membrane (Millipore). After incubation with primary and secondary antibodies, the blots were developed by chemiluminescence using Immobilon Classico Western HRP substrate (Merck Millipore) or enhanced chemiluminescence reagents (GE Healthcare). Densitometry was performed with ImageJ software. For western blotting of weak signal antibodies, primary antibody was diluted with SignalBoost Immunoreaction Enhancer Kit (Merck Millipore, 407207) and blots were developed with Luminata Crescendo Western HRP substrate (Merck Millipore).

GBM cells were lysed with a commercially available buffer containing Triton X-100 and protease/phosphatase inhibitors (both from Cell Signaling Technology, Frankfurt am Main, Germany). Cell debris was removed by centrifugation and the lysates were incubated with an SDS-Loading buffer containing 4% glycerin, 0.8% SDS, 1.6% beta-mercaptoethanol and 0.04% bromophenol blue (all from Carl Roth). Samples were separated by SDS-PAGE followed by transfer to Immobilon-P (Merck Millipore) or Roti^®-Fluoro (Carl Roth) PVDF membranes. The membranes were incubated with the following primary antibodies: anti-Catenin D1, anti-CD44, anti-CD99 (from Cell Signaling Technology), anti-MT1-MMP and anti-PLOD2 (from Proteintech) overnight at 4 °C. Secondary reactions were performed for 1h at room temperature using HRP-, AlexaFluor^®488- or AlexaFluor^®647-coupled antibodies (all from Cell Signaling Technology). All antibodies were diluted as recommended by the respective manufacturer using the SignalBoost™ Immunoreaction Enhancer Kit (Merck Millipore). Signal detection was performed on a ChemoStar imaging system (Intas Science Imaging, Göttingen, Germany).