Anti actb

SDS-PAGE was done by using Cleaver Scientific (Rugby, UK) omniPAGE system. Proteins were transferred onto Millipore 0.45 μm nitrocellulose membrane. Immunoblotting was performed using TBS Tween (0.1%), containing 5% non-fat dry milk for blocking membrane and 1% non-fat dry milk for antibody solutions. Loading was controlled by developing membranes for β-actin or GAPDH. The following antibodies were applied: Rabbit PolyAb Anti-PARPI (Proteintech^®, Rosemont, IL, USA, 13371-1-AP), Rabbit PolyAb Anti-RIPK1 (Proteintech^®, 17519-1-AP), Rabbit PolyAb Anti-RIPK3 (Proteintech^®, 17563-1-AP), Anti-P-c-Jun (Cell Signaling, Danvers, MA, USA, 9261S), Anti-c-Jun (Cell Signaling, 9165S), and Anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA, 6C5). Rabbit PolyAb Anti-ACTB (Proteintech^®, 20536-1-AP), antiHRP-conjugated secondary antibodies: HRP-Goat Anti-Rabbit IgG (Proteintech^®, 00001-2), HRP-linked Anti-Mouse IgG (Proteintech^®, 7076S). The bands were visualized using a chemiluminescence detection kit (Thermo Scientific™, 32,106) and VWR™ (Radnor, PA, USA) Imager Chemi Premium gel documentation system with VWR™ Image Capture Software (version: 1.6.1.0). For densitometry analysis, Western blot data were acquired using ImageJ software bundled with 64-bit Java 1.8.0_172.

Total protein was extracted from cells using a cell extraction buffer [50 mM Tris-HCl (pH 8.0), 4 M urea, and 1% Triton X-100] containing a protease inhibitor cocktail (Roche Diagnostics, catalogue number 04693132001). The samples were subsequently resolved using SDS-PAGE and analyzed by western blotting. The following antibodies were used: anti-H3 (CTS, #4499, 1:1,000), anti-γH2AX (CTS, #9718, 1:1,000), anti-H3K27cr (RevMab Biosciences, 31-1287-00, 1:800), anti-SIRT6 (Proteintech, #13572-1-AP, 1:1,000), anti-ACTB (Proteintech, #20536-1-AP, 1:5,000), anti-rabbit IgG (KPL, 074-1506, 1:5,000), and anti-mouse IgG (KPL, 074-1806, 1:5,000). The intensity of image was measured by Image J2 software (https://imagej.net/Fiji/Downloads).

The total cell protein was extracted using a kit (TransGen Biotech, China),
and the protein concentration was detected using a BCA kit (TransGen
Biotech). A 10 % separating gel and 5 % stacking gel were prepared for
electrophoresis. The band was excised and transferred to the membrane,
following which it was incubated overnight at 4  ${}^{\circ }$ C with the
primary antibodies, namely anti-Lin28B (1 : 1000, Abcam, UK) and anti-ACTB
(1 : 5000, Proteintech). The membrane was then incubated with an
enzyme-conjugated secondary antibody for 2 h at 37  ${}^{\circ }$ C. The bands
were detected using an ELC luminescence kit (Beyotime, Jiangsu, China).