Ecl select wb detection reagent

Native H1N1pdm viruses were detected using antisera from rabbits hyper-immunized with NA-VLPs, followed by goat anti-Rabbit IgG HRP-conjugated secondary antibody (Thermo Fisher). HRP-conjugated anti-FLAG M2 monoclonal antibody (Merck) was used for the detection of FLAG-tagged HA and NA proteins on VLPs. For western blotting, samples were denatured by heating at 95°C for 10 min in LDS sample buffer (Invitrogen) with DTT (100 mM) and were resolved in SDS-PAGE, followed by electroblotting onto PVDF membrane. The PVDF membranes were hybridized with the corresponding antibodies and detected using ECL Select WB Detection Reagent (GE Healthcare). The relative amounts of protein in each band were determined by densitometry using ImageJ (NIH).

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).

Cells were lysed with RIPA buffer supplemented with protease and phosphatase inhibitors. Protein samples were subjected to SDS-PAGE and transferred to PVDF membranes (Hybond^TM, Fisher Scientific). The antibodies used are provided in Supplementary Table S4. Immunoblots were revealed using the ECL Select WB Detection Reagent (GE Healthcare) and an Alliance LD2 system (UVITEC). WB were performed in at least three independent biological replicates; representative data are shown.