Anti rnf8

For IF experiments, cells plated on coverslips were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 10 min and blocked with 5% goat serum for 1 h. The coverslips were incubated sequentially with primary antibody at 4 °C overnight and fluorescence-labeled secondary antibody for 1 h at room temperature. For IF-FISH, the coverslips were then hybridized with PNA probe (TelG-Cy3, Panagene). Coverslips were stained with DAPI (Vector Laboratories) and visualized using fluorescence microscopy.
Antibodies used are as follows: anti-cGAS (1:100, CST), anti-53BP1 (1:2,000, NB100-304, Novus), MDC1 (1:400, ab11171, Abcam), anti-RNF8 (1:100, Santa Cruz), anti-CDK1 (1:200, Abcam), anti-γH2AX antibody (1:400, CST), anti-γH2AX antibody (1:400, ab26350, Abcam).

Protein expression was detected by western blot analysis as described previously
[19] (link). Briefly, RIPA buffer with 1× protease inhibitor cocktail (Roche, Basel, Switzerland) was used to lyse cells. Equal amounts of protein were separated by SDS‒PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, USA). Membranes were blocked with 5% nonfat powdered milk (Sangon Biotech, Shanghai, China) in Tris-buffered saline containing 0.1% Tween-20 (TBST), incubated with the primary antibodies including anti-RNF8 (Santa Cruz Biotech, Santa Cruz, USA), anti-phospho-β-catenin (Cell Signaling Technology, Beverly, USA) and anti-β-actin (Santa Cruz Biotech), followed by incubation with the corresponding secondary antibodies (HRP-conjugated anti-mouse IgG or anti-rabbit IgG (Fc); ZSGB-Bio, Guangzhou, China). An epithelial-mesenchymal transition antibody sample kit (Cell Signaling Technology) was used to detect EMT-related proteins. The immunoreactive bands were visualized using an ECL detection system (Millipore). All experiments were repeated with three independent replicates.

The commercial antibodies used for Western blot analysis are as follows: anti-Twist1 (1:1000, sc-81417, Santa Cruz Biotechnology), anti-USP13 (1:3000, ab109264, Abcam), anti-RNF8 (1:1000, sc-271462, Santa Cruz Biotechnology), anti-FBXL14 (1:1000, HPA053889, Sigma-Aldrich), anti-βTrcp1 (1:1000, sc-390629, Santa Cruz Biotechnology), anti-E-cadherin (1:1000, sc-8426, Santa Cruz Biotechnology), anti-N-cadherin (1:1000, sc-59987, Santa Cruz Biotechnology), anti-Vimentin (1:1000, sc-6260, Santa Cruz Biotechnology), anti-MMP9 (1:1000, sc-21733, Santa Cruz Biotechnology), anti-Ubiquitin (1:1000, sc-8017, Santa Cruz Biotechnology), anti-Flag (1:3000, GTX115043, GeneTex), anti-GFP (1:3000, GTX113617, GeneTex), anti-HA (1:3000, GTX115044, GeneTex), anti-c-Myc (1:3000, PLA0001, Sigma), anti-GST (1:3000, sc-138, Santa Cruz Biotechnology), anti-GAPDH (1:3000, GTX627408, GeneTex), anti-tubulin (1:3000, bs-0715R, Bioss), and anti-Ubiquitin K48-specific antibody (1:1000, ZRB2150, Sigma-Aldrich). anti-Flag Affinity Gel (B23102), Poly Flag Peptide (B23111) and Protein A/G mix magnetic beads (B23202) were obtained from Bimake Chemicals. Cycloheximide (S7418) and MG132 (S2619) were obtained from Selleck Chemicals. TGF-β1 (100–21) was obtained from Peprotech.