Subcellular fractionation protocol

In order to separate nuclear and cytoplasmatic fractions from TTR KO hippocampal neurons cellular extracts (13 DIV), cultured neurons were homogenized in buffer containing 10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT and 0.05% NP40 at pH 7.9, supplemented with 1× protease inhibitors mixture (GE Healthcare). Neurons were extracted in 80 µl of chilled supplemented buffer, using a cell scraper, and kept on ice for 10 min. Then, neurons were centrifuged at 720 × g, 4°C for 5 min. The pellet containing the nuclear fraction was resuspended in TBS (Tris-buffered saline) with 0.1% SDS, while the supernatant was centrifuged again for 10 000 × g for 5 min. The obtained supernatant contained mostly the cytoplasmatic fraction (with some membranes). Both fractions were then sonicated on ice, which is particularly important for nuclear fractions, in order to shear genomic DNA and homogenize the lysate. Western blot confirmed nuclear fraction enrichment by Histone H2Ax in opposition to GAPDH. This protocol was based on Abcam ‘Nuclear Extraction and fractionation protocol’ and ‘Subcellular fractionation protocol’.