TRABID deubiquitinase overexpression enables synthetic lethality to PARP inhibitor via prolonged retention of 53BP1 at DSB sites

Published: 27 February 2023| Version 1 | DOI: 10.17632/n9txt6y5cj.1
Jian Ma


Here, we demonstrate that TRABID deubiquitinase (DUB) binds to 53BP1 at endogenous level and regulates 53BP1 retention at DSB sites. TRABID deubiquitinates K29-linked polyubiquitination of 53BP1 mediated by E3 ubiquitin ligase SPOP and prevents 53BP1 dissociation from DSBs, consequently inducing HR defects and chromosomal instability. Prostate cancer cells with TRABID overexpression exhibit a high sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors. This dataset include all the raw data of western blot from this project.


Steps to reproduce

Cells were harvested and lysed by IP buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate and 1% protease inhibitor cocktails) on ice for more than 15 min. Cell lysate was centrifuged for 15 min at 13,000 rpm at 4°C, and the supernatant was incubated with primary antibodies and protein A/G agarose beads (Thermo Fisher Scientific) with rotating at 4°C overnight. The next day, the pellet was washed at least six times with 1×IP buffer on ice, and then subjected to western blotting analysis. For ubiquitination using Flag-conjugated agarose beads or Flag primary antibody plus protein A/G beads, 293T cells were transfected with plasmids for HA-Ub, Flag-53BP1 and other indicated constructs. Cells were harvested and lysed with lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate and 1× protease inhibitor cocktail (PIC)). The lysate was subjected to co-immunoprecipitation using anti-Flag-conjugated agarose beads or Flag primary antibody plus protein A/G beads. For ubiquitination using nickel-nitrilotriacetic acid (Ni-NTA) beads (QIAGEN), cells were transfected with His-ubiquitin and indicated constructs for 42 h. Subsequently, cells were lysed with buffer A (6 M guanidine-HCl, 0.1 M Na2HPO4/NaH2PO4, and 10 mM imidazole [pH 8.0]) and sonicated for 15 seconds. After incubating with nickel-nitrilotriacetic acid (Ni-NTA) beads (QIAGEN) for 3 h at room temperature, the proteins were washed twice with buffer A, twice with buffer A/TI (1 volume buffer A and 3 volumes buffer TI), and one time with buffer TI (25 mM Tris-HCl and 20 mM imidazole [pH 6.8]). The pull-down proteins were denatured by boiling at 95 °C for 5 min and separated by SDS-PAGE for immunoblotting.


Western Blot