Ab4080

Total protein was extracted using Radio Immunoprecipitation Assay (RIPA) lysis buffer (BYL40825; JRDUN Biotechnology, Shanghai, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, USA). The membrane was blocked with 5% skim milk and then incubated with primary antibodies against p53 (2524; Cell Signaling Technology, Danvers, USA), p21 (2947; Cell Signaling Technology), USP7 (ab4080; Abcam, Cambridge, USA,), or GAPDH (5174; Cell Signaling Technology), followed by incubation with the corresponding horse radish peroxidase (HRP)-conjugated secondary antibodies (A0208 and A0216; Beyotime). The content of protein was detected by using enhanced chemiluminescence reagents (Bio-Rad).

The transfected cells were lysed in lysate buffer (mixture of 50 mmol/L Tris ‐ HCl (pH 7.4), 150 mmol/L NaCl, 10% glycerol, 1 mmol/L EDTA, 0.5% NP‐40 and protease inhibitor) and centrifuged to move cell debris. Cleared cell lysate was incubated with 1 μg anti‐HA (ab9110, 1:70, Abcam, Cambridge, UK), myc (ab32072, 1:100, Abcam, Cambridge, UK), USP7 (ab109109, 1:1000, Abcam, Cambridge, UK), KDM6B (ab38113, 1:100, Abcam, Cambridge, UK) or anti‐FLAG antibody (ab205606, 1:1000, Abcam, Cambridge, UK) and 15 μL protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 hours. After extensive washing, the beads were boiled at 100℃ for 5 minutes. Proteins were resolved by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Millipore, Temecula, CA, USA), followed by immunoblotting. To detect endogenous protein interactions, cells were lysed in ice‐cold lysis buffer. Cleared cell lysates were incubated with 5 μg anti‐USP7 antibody (ab4080, 1:1000, Abcam, Cambridge, UK) and 20 μL protein A/G beads at 4°C overnight. The anti‐USP7 antibody was used to detect the endogenous KDM6B.

Antibodies used in this study includeUSP7(Western blot: #4833, CST; ab4080, Abcam; Immunohistochemistry: NB100-513, NOVUS), SAMHD1 (Western blot:#12361, CST; IP: ab245389, Abcam; Immunohistochemistry/Immunofluorescence: ab128107, Abcam), CtIP (Western blot: #9201, CST;Immunofluorescence:NB100-79810), β-actin (AC004, ABclonal), Flag (SG4110-16, Shanghai Genomics Technology), Myc (SG4110-18, Shanghai Genomics Technology), HA (#3724, CST), Ubiquitin (#3933S, CST), PARP (#9532, CST),Cleaved PARP (#5625, CST), Caspase3 (#9662, CST), Cleaved Caspase3 (#9664, CST), Phospho-Histone H2A.X (Ser139) (#9718, CST; #80312, CST).
CHX (S7418), P5091 (S7132), Cisplatin (S1166), Doxorubicin (Adriamycin) HCl (S1208), and MG132 (S2619) were purchased from Selleck. PI (propidium iodide, ST511) and NAC (S0077) were from Beyotime. IPTG (I6758) were from Sigma-Aldrich.

Western blotting analysis was performed on all individual samples in the identification cohort. For sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 20 μg of protein was separated by gel electrophoresis on 10% Mini-PROTEAN TGX Precast Gels (#456-1084; Bio-Rad Laboratories, Shanghai, China) and transferred to polyvinylidene fluoride membranes. The membranes were then incubated at 4°C overnight with primary anti-WDR1 antibody (1:1000, ab228738, Abcam, Shanghai, China), primary anti-USP7 antibody (1:1000, ab4080, Abcam, Shanghai, China) or primary anti-β-Catenin antibody (1:5000, ab32572, Abcam, Shanghai, China), followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1:1000, GE Healthcare Biosciences, NY, USA) for 1 h at room temperature. β-actin was used as a control for protein loading, and was detected using a mouse monoclonal anti-β-actin antibody (1:1000, ab5694, Abcam, Shanghai, China). The signal was visualised using an enhanced chemiluminescence agent (GE Healthcare Biosciences, NY, USA), and quantitative analysis of the protein bands was performed by using ImageJ software (NIH Image).