Nuclear extraction and fractionation protocol

The effect of LCS-1269 on HDAC expression was assessed on HeLa TI cells using Western blotting. Cells were incubated with LCS-1269 at concentrations of 2 and 4 μM for 24 h in 60 mm Petri dishes. The nuclear fraction of the cells was obtained according to the Abcam Nuclear extraction and fractionation protocol. Nuclear proteins were separated by 10% PAGE and transferred to 0.22 μm nitrocellulose membranes; transfer conditions for proteins up to 25 kDa were 40 min at 100 mA, for proteins from 25 to 100 kDa—40 min at 100 mA and 20 min at 200 mA. Blocking with BSA, primary and secondary antibodies was performed as described above. The study used Abcam antibodies to HDAC1 (ab53091), histone H4 (load control, ab10158) and secondary anti-rabbit antibodies (ab6721). Protein detection and densitometric analysis were performed as described above. Experiments were performed three times.

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’.