FIGURE 1.

Bora is degraded in a proteasome-dependent manner following DNA damage. A–C, 293T cells were transfected with a construct encoding FLAG-Bora. Cells were left untreated or treated with UV (20 J/m²), camptothecin (CPT, 1 μm), irradiation (IR, 10 gray). Cells were collected at indicated time (A) or 1 h (B and C) later and examined by immunoblot analysis. The pChk1s317 blot served as a positive control for UV treatment. DMSO, dimethyl sulfoxide. D, 293T cells were U- irradiated, collected at the indicated time, and then endogenous Bora levels were examined with Bora antibody. E, 293T cells were UV-irradiated and fixed at the indicated time. Cell cycle distribution was determined by FACS. The results represent the mean values from three independent experiments. Error bars represent S.E. #, p > 0.05. F, 293T cells were harvested 2 h post-UV radiation, mRNA was extracted, and Bora levels were determined by quantitative RT-PCR. Error bars represent S.E. of n = 3. #, p > 0.05. G, 293T cells were pretreated with DMSO or 50 μm MG132 for 3 h and then treated with UV radiation. 2 h later, cells were harvested, and Bora levels were examined by Western blot analysis. H, 293T cells were pretreated with DMSO or 40 μm MG132 for 3 h and then treated with 20 J/m² of UV radiation. Endogenous Bora ubiquitination (Ub) was then examined by immunoblot analysis. IP, immunoprecipitation.