Fig. 2.

TRIM25 binds CHKα and polyubiquitylates CHKα at K195 for its degradation. (A) Endogenous TRIM25 in U87 cells was immunoprecipitated. Immunoblotting analyses were performed with the indicated antibodies. (B) Bacterially purified His-TRIM25 and GST-CHKα were mixed. A GST pulldown assay was performed. Immunoblotting analyses were performed with the indicated antibodies. (C) A schematic shows WT and truncated TRIM25 (Top). Flag-CHKα was coexpressed with or without the indicated HA-TRIM25 proteins in HEK293T cells in the presence of MG132 (10 μM) for 10 h. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies (Bottom). RING, RING-finger domain; B1, the 1st B-box domain; B2, the 2nd B-box domain; middle, middle region; PRY, PRY domain; SPRY, SPRY domain. (D) U87 cells with or without expression of the indicated HA-TRIM25 proteins were analyzed by immunoblotting analyses with the indicated antibodies. Arrow indicates TRIM25-∆PRY-SPRY. (E) U87 (Left) and U251 (Right) cells were stably transfected with vectors expressing the indicated shRNA. Immunoblotting analyses were performed with the indicated antibodies. (F) An in vitro ubiquitylation assay was performed by incubating purified GST-CHKα with the indicated purified HA-TRIM25 proteins. Immunoblotting analyses were performed with the indicated antibodies. (G) U87 cells were transfected the indicated plasmids. The cells were treated with MG132 (10 μM) for 10 h. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. (H) HEK293T cells expressing His-ubiquitin, HA-TRIM25, and the indicated Flag-CHKα proteins were treated with MG132 (10 μM) for 10 h. Immunoprecipitation and immunoblotting were performed with the indicated antibodies. (I) U87 cells expressing Flag-CHKα or Flag-CHKα K195R were treated with CHX (100 μg/mL) for indicated periods of time. Immunoblotting analyses were performed with the indicated antibodies (Top). The expression levels of CHKα were quantified (Bottom).