Autoradiographic film

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).

Frozen lung tissue was homogenized on ice using a rotor blender (Fisher) on ice in Tissue protein extraction reagent (T-Per from Sigma). This was supplemented with phosphatase and protease inhibitors (Halt; Sigma). Homogenates were then centrifuged for 15 min at 4°C, and the supernatants were collected and frozen at −80°C until required. The protein concentration was established using a BCA technique (Thermo Scientific), and 30–40 μg of protein were then separated by electrophoresis on a Bis-Tris NuPage gel. Proteins were then transferred to PVDF Immobilon and transfer was confirmed with Ponceau red stain. The blot was blocked at room temperature for 1–2 h in 5% nonfat milk in Tris-buffered saline containing 0.05% Tween-20. Membranes were then incubated overnight at 4°C with primary antibody diluted accordingly in 5% milk/TBS-T. These were subsequently washed using TBS-T and then incubated with secondary antibody for 1–2 h at room temperature. The antibody labeling was visualized using enhanced chemiluminscence (ECL; Amersham) with exposure to autoradiographic film (GE Healthcare).

Total intracellular RNA was isolated using an RNeasy kit (Qiagen) according to the manufacturer’s instructions. Northern blot transfer, probe preparation, and probe hybridization were performed as described previously^{59 (link)}. Negative and positive sense ³²P-labeled CAT-specific riboprobes were synthesized with T7 RNA polymerase as described previously^{60 (link)}. The CAT1 mRNA and antigenome RNA on Northern blots were identified by migrating RNA alongside a molecular weight ladder (Dynamarker prestain marker for RNA high). Following hybridization, the membrane was exposed to autoradiographic film (GE Healthcare), the film was aligned to the blot with the colored markers, and the positions of the markers were marked onto the film, allowing us to confirm the sizes of the RNAs. Subsequent Northern blots were analyzed by phosphorimager analysis: data acquisition was made on phosphorimager (Personal Molecular Imager, Bio-Rad) using the associated software Quantity one 1-D (Bio-Rad). The background signal was subtracted from each sample value, and the resulting value was then normalized to the corresponding RNA signal of the FLAG-tagged WT L.