Figure 5.

USP20 phosphorylation by ATR is important for its stabilization and checkpoint activation. (A) Cells treated with MG132 or caffeine as indicated for 1 h were left untreated or treated with UV. Cell lysates were subjected to immunoprecipitation with anti-USP20 antibody. The immunoprecipitates were then blotted with the indicated antibodies. (B) Cells were left untreated or treated with UV or HU. After 1 h, USP20 was immunoprecipitated with anti-USP20 antibody and immunoblotted with phospho-SQ/TQ antibody. (C) Cells were pretreated with Dimethyl sulfoxide (DMSO) or 3 mM caffeine. After 2-h incubation, cells were left untreated or treated with UV (30 J/m²). After an additional 1 h, USP20 was immunoprecipitated with anti-USP20 antibody and immunoblotted with phospho-SQ/TQ antibody. (D) Cells stably expressing control shRNA or ATR shRNA were left untreated or treated with UV. After 1 h, USP20 was immunoprecipitated with anti-USP20 antibody and immunoblotted with phospho-SQ/TQ antibody. (E and F) Cells transfected with indicated constructs were left untreated or treated with UV (30 J/m²). After 1 h, HA-USP20 was immunoprecipitated with anti-HA antibody and immunoblotted with phospho-SQ/TQ antibody. (B–F) The immunoprecipitated USP20 loading levels were equalized. (G) Cells stably expressing USP20 shRNA were transfected with shRNA resistant HA-USP20 WT or USP20-4mut mutant. After 48 h, cells were treated with MG132. 2 h later, cells were left untreated or treated with UV (30 J/m²) and after an additional 1 h, cell lysates were subjected to immunoprecipitation with anti-HA antibody. The immunoprecipitates were then blotted with the indicated antibodies. (H) Cells from (G) were left untreated or treated with UV (30 J/m²) or HU (10 mM). Cells were lysed and cell lysates were then blotted with the indicated antibodies. (I and J) Cells expressing the indicated constructs were pretreated for 15 min with CHX (0.1 mg/ml) followed by UV (50 J/m²) treatment. Cells harvested at the indicated times were lysed and cell lysates were then blotted with the indicated antibodies. (K) Cells stably expressing control or USP20 shRNA were stably transfected with empty vector, shRNA-resistant HA-USP20WT or HA-USP20-4mut mutant. Cells were treated as indicated. After 2 weeks, cell viability was determined by colony-formation assay. Error bars represents the SEM of three independent experiments. (L) Cells the same as (K) were left untreated or treated with HU (10 mM) or UV (30 J/m²). After 2 h, cells were incubated with 20 μM BrdU, 30 min later, cells were fixed and stained with anti-BrdU antibody. Error bar represents the SEM of three independent experiments. **P < 0.01.