Fig. 4

DNA damage-induced BRE-USP7 interaction stabilizes CDC25A. a Ubiquitylation of FLAG-CDC25A in cells harvested 4 h after 6 Gy IR in absence (lanes 1–4) or presence (lanes 5, 6) of exogenous BRE. CDC25A was immuno-precipitated using anti-FLAG antibody (lanes 1, 2, 5, 6), IgG was used as control (lanes 3, 4). b MCF7 cells stably expressing CRE-inducible HA-BRE were transfected with either non-specific (NS, lanes 1–2 and 7–8) or two independent USP7-specific (USP7 #1, lanes 3, 4, 9, 10 and USP7 #2, lanes 5, 6, 11, 12) siRNAs along with or without plasmids expressing CRE and irradiated with 6 Gy IR. Western blot shows the abundance of endogenous CDC25A in non-IR cells and 4 h after irradiation. HDM2 was used as a control for USP7 knockdown. c FLAG-CDC25A ubiquitylation in cells harvested 4 h after 6 Gy IR in absence (lanes 1–3) or in presence of exogenous USP7 (lanes 4, 5) or catalytic inactive USP7 (USP7-CS; lanes 6, 7). Anti-FLAG antibody (lanes 2–7) was used to pull down CDC25A, IgG was used as control (lane 1). HDM2 was used as a control for USP7 activity. d Co-immunoprecipitation of FLAG-CDC25A, BRE, and endogenous USP7 from MCF7 cells using anti-BRE antibody (lanes 1–4) and IgG antibody (lanes 5–6); 5 μM MG132 was added after irradiation to increase CDC25A level and harvested after 2 h. e Model depicting the interaction of BRE, CDC25A, and USP7. In presence of DNA breaks, BRE interacts with USP7 and then BRE, USP7, and CDC25A complex is formed. For simplicity, MERIT40 is not included in the model. f Co-immunoprecipitation of USP7 and different deletion mutants of BRE tagged with HA-epitope from irradiated HEK293 cells using USP7 antibody. Top panel shows the schematic representation of different deletion mutants of BRE. Numbers in the top panel mark different USP7 binding motifs. Stars indicate different HA-BRE proteins. g Western blots showing IR-induced degradation of CDC25A after expressing HA-BRE or HA-BRE mutant defective in USP7 binding (FLΔ123). GAPDH was used as a loading control for all blots. Histogram represents mean ± s.d. values of relative band intensities. P values are calculated using the paired two-tailed t-test and presented in Supplementary Table 2