Anti histone h3k36me3

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).