Nupage novex bis tris protein gradient polyacrylamide gels

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).

Total extracts were prepared as previously described [55 (link)]. Briefly, to obtain total protein extracts, cells were washed once with PBS (1×) and total cell lysates were prepared using RIPA [56 (link),57 (link)]. The samples were centrifuged at 12,000 rpm for 20 min at +4 °C and supernatants containing the total extracts were recovered. Proteins were separated on 4–12% NuPAGE Novex Bis-Tris protein gradient polyacrylamide gels (Thermo Fisher Scientific) and blotted onto nitrocellulose. Membranes were blocked with 5% milk (BioRad) and then incubated with the following antibodies: anti-GPX4 (1:500, ab41787), anti-GAPDH-HRP-conjugated (1:1000, sc-47724). Peroxidase AffiniPure Sheep Anti-Mouse IgG, Peroxidase AffiniPure Donkey Anti-Rabbit IgG, Peroxidase AffiniPure Donkey Anti-Goat IgG (1:10,000, Jackson ImmunoResearch Europe Ltd. Cambridge House) secondary antibodies were used. Signals were detected using the WESTARɳ2.0 ECL substrates for Western blotting (XLS070,0250, Cyanagen, Bologna, Italy) and acquired using a Uvitec Alliance Mini HD9 (Uvitec, Cambridge, UK).