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