Sc 74559

Primary antibodies used in this study were as follows: mouse anti-USP4 (66822; Proteintech1:1000 dilution for WB, 1:300 dilution for IHC); mouse anti-BRCA1 (sc-6954; Santa Cruz; 1:300 dilution for WB); rabbit anti-BRCA1(ab16780; Abcam;1:400 dilution for IHC); rabbit anti-BRCA1 (ab191042; Abcam;1:1000 dilution for WB); rabbit anti-BARD1 (NB100; Novus Biologicals; 1:1000 dilution for WB); mouse anti-BARD1 (sc-74559; Santa Cruz; 1:500 dilution for WB); mouse anti-Flag (F3165; Sigma; 1:5000 dilution for WB); rabbit anti-HA (3924 S; Cell Signaling Technology; 1:1000 dilution for WB); mouse anti-Myc (M4439; Sigma;1:5000 dilution for WB); mouse anti-ubiquitin (3936 S; Cell Signaling Technology; 1:1000 dilution for WB); mouse anti-α-His (2366 S; Cell Signaling Technology; 1:1000 dilution for WB); mouse anti-α-Tubulin (3873 S; Cell Signaling Technology; 1:3000 dilution for WB), rabbit anti-β-actin (AC026; ABclonal; 1:1000 dilution for WB); rabbit anti-GAPDH(5174 S; Cell Signaling Technology; 1:5000 dilution for WB). For WB, western blots were detected and analyzed using ChemiDoc Imaging system (Bio-Rad). The original WB blots were included in the supplementary information.

Cells were lysed in RIPA lysis buffer (Beotime, China) supplemented with protease and phosphatase inhibitors (Life Technologies, USA). Protein samples were subjected to 8% SDS-PAGE and transferred to PVDF membranes (Bio-Rad, USA). Membranes were then blocked with 5% non-fat milk in 0.1% TBST buffer overnight at 4 °C. The membranes were subsequently incubated with BRAD1 (sc-74559, Santa Cruz, 1:1000), BRCA1 (OP92, Calbiochem, 1:500), p-BRCA1 (sc-24512, Santa Cruz, 1:500), CDK1 (9116, CST, 1:1000), CDK12 (11973, CST, 1:1000), Lig4 (ab26039, Abcam, 1:1000), AKT (4685, CST, 1:1000), P-AKT (2965, CST, 1:1000), H2AX/γH2AX antibody (2577/2599, CST, 1:1000). The protein–antibody complex was detected using HRP-conjugated secondary antibodies and enhanced chemiluminescence (Pierce). The band intensity was quantified using ImageJ software (NIH, Bethesda). Uncropped scans of western blots presented in the main figures are provided in Supplementary Figs 9–13.