Nuclear and Cytoplasmic Protein Extraction

HeyA8 and ES-2 cells were processed for nuclear and cytoplasmic extracts. Cells were scraped, resuspended in hypotonic lysis buffer 10mM HEPES pH7.9, 10mM KCl, 0.1mM EDTA, protease inhibitors (P8849, Sigma-Aldrich) and phosphatase inhibitor cocktails 2 and 3 (P0044, P5726, Sigma-Aldrich) and incubated on ice for 20 min. After the addition of 0.25% Igepal-630 (NP40) (Sigma-Aldrich), samples were centrifuged at 3000 rpm for 5 min and supernatants containing the cytoplasmic extracts were recovered. Nuclear pellets were resuspended in 20mM HEPES pH7.9, 0.4M NaCl, 1mM EDTA with protease and phosphatase inhibitors. After three cycles of vortex and ice, samples were centrifuged at 12000 rpm for 20 min and the supernatants containing the nuclear extracts were collected.
Proteins were separated on 4–12% NuPAGE Novex Bis-Tris Protein gradient polyacrylamide gels (Thermo Fisher Scientific) and blotted onto nitrocellulose. Membranes were quenched with 5% blotto (BioRad) [42 (link)] and the proteins detected with: rabbit anti-ZNF521 (EHZF S15 sc-84808, Santa Cruz Biotechnology, DBA, Milan, Italy) at 1:1000, rabbit anti- HDAC1 (H3284, Sigma) 1:10000. Secondary rabbit HRP antibody at 1:2000 (65–6120 Thermo Fisher Scientific) was detected by the ImmunoCruz Western blotting luminal reagent (sc-2004, Santa Cruz, Biotechnology) and exposure to autoradiographic film (GE Healthcare, Milan, Italy).