Amersham protran 0.45 μm nitrocellulose

The hiNPCs were washed with PBS and resuspended in 100 μl of extraction buffer [10 mM HEPES pH 8, 10 mM MgCl₂, 0.1 mM EDTA pH 8, 0.1 mM DTT and halt protease and phosphatase inhibitor cocktail (Life Technologies)]. Samples were centrifuged at 5000 rpm for 10 min at 4°C to remove the cytosolic fraction. Nuclear pellets were resuspended in 0.2 N HCl and put in rotation at 4°C overnight. After centrifugation at 4000 rpm for 10 min at 4°C, supernatants containing nuclear proteins were recovered. Proteins were quantified by Bradford Protein Assay Kit (Sigma-Aldrich). Protein samples were separated by 4–12% Bis–Tris Protein Gels (Thermo Fisher) and transferred on an Amersham™ Protran™ 0.45 μm nitrocellulose (GE-Healthcare) membrane. Membranes were blocked with 5% w/v non-fat dried milk and incubated with the following primary antibodies: anti-CHD8 (NB100-60417, Novus Biologicals) (1:1000), anti-HSP90 (4874S, Cell Signaling Tech.) (1:5000), anti-histone H3 (1:1.000) (4499, Cell Signaling Tech.) and anti-histone H3K36me3 (1:1.000) (Ab9050, Abcam). Proteins were detected using horseradish peroxidase-conjugated secondary antibodies anti-rabbit IgG 1:7500 (GTX213110-01, GeneTex) and visualized by ECL Select WB detection reagent (GE Healthcare) following the manufacturer's instructions. Signal quantification was performed with Imagelab software (BIORAD, version 5.2.1).

Samples were run on gels consisting of a 4% w/v acrylamide stacking gel (4% w/v acrylamide, 0.125 M Tris-HCl (pH 6.8), 0.2% v/v tetramethylethylenediamine, and 0.08% w/v ammonium persulfate (APS)) and 10% w/v acrylamide separating gel (10% w/v acrylamide, 0.375 M Bis-Tris (pH 6.8), 1% v/v tetramethylethylenediamine, and 0.05% w/v APS) in MOPS buffer (50 mM MOPS, 50 mM Tris, 1 mM EDTA, 0.1% w/v SDS) at 90–120 V. Proteins were electrophoretically transferred onto nitrocellulose membranes (Amersham Protran 0.45 μm nitrocellulose; GE Healthcare) at 90 V for 90 min on ice in transfer buffer (48 mM Tris/HCl, 39 mM glycine, 20% v/v methanol). Transferred membranes were blocked with 5% w/v nonfat dry milk dissolved in TBS-T (20 mM Tris/HCl (pH 7.5), 150 mM NaCl, and 0.1% v/v Tween 20) at room temperature for 1 h. Membranes were then incubated with primary antibodies overnight at 4°C. After washing membranes in TBS-T 3 × 15 min, membranes were incubated with secondary antibodies at room temperature for 1 h. After washing membranes in TBS-T 3 × 15 min, membranes were scanned using LI-COR Odyssey CLx. Protein band intensities were analyzed using LI-COR Image Studio Lite software.