Figure 4.

USP20 is regulated by HERC2 following DNA damage or replication stress. (A) Cells treated with MG132. After 1 h, cells were left untreated or treated with HU (10 mM) or UV (30 J/m²). After 2 h, USP20 was immunoprecipitated and immunoblotted with the indicated antibodies. (B and C) HEK293T cell lysates were subjected to immunoprecipitation with control IgG, anti-USP20 (B) or anti-HERC2 (C) antibodies. The immunoprecipitates were then blotted with the indicated antibodies. (D) Cells stably expressing control shRNA or HERC2 shRNAs were lysed and cell lysates were then blotted with the indicated antibodies. (E) Cells stably expressing control shRNA or HERC2 shRNA were treated with CHX (0.1 mg/ml), and harvested at the indicated times. Cells were lysed and cell lysates were then blotted with the indicated antibodies. Right panel: quantification of the USP20 protein levels relative to β-actin. (F) Cells stably expressing Ctrl or HERC2 shRNAs were treated with MG132 for 4 h before harvest. USP20 was immunoprecipitated and immunoblotted with the indicated antibodies. (G) FLAG-HERC2-HECT-WT and CA mutant were purified from HEK-293T cells and incubated with E1, E2, ubiquitin (HA-Ub), ATP and GST-UPS20 or in the absence of the indicated reagents. Ubiquitinated products were detected by immunoblot with anti-HA antibody. (H) Cells treated with MG132 for 1 h were left untreated or treated with UV and cells were harvested at the indicated times. Cell lysates were subjected to immunoprecipitation with anti-USP20 antibody. The immunoprecipitates were then blotted with the indicated antibodies. (I) Cells stably expressing control shRNA or HERC2 shRNA were left untreated or treated with UV. Cells were lysed and cell lysates were then blotted with the indicated antibodies.