FIGURE 1.

USP10 interacts with MSH2. A and B, endogenous USP10 and MSH2 interact with each other. A, 293T cells and MEFs were lysed and immunoprecipitated (IP-ed) with either anti-IgG or anti-USP10 antibodies followed by an anti-MSH2 Western blotting analysis (upper panel). The blot was stripped and reprobed with anti-USP10 antibodies (lower panel). B, HeLa cells and MEFs were lysed and IP-ed with either anti-IgG or anti-MSH2 antibodies followed by an anti-USP10 Western blotting analysis (upper panel). The blot was stripped and reprobed with anti-MSH2 antibodies (lower panel). C, diagrams of the domain structure of MSH2 and the deletion constructs of MSH2. D, USP10 physically interacts with MSH2, and the N terminus of MSH2 binds to USP10. Escherichia coli lysates harboring His-USP10 were incubated with bacterially-purified GST, GST-MSH2, GST-MSH2-(1–378), GST-MSH2-(200–700), or GST-MSH2-(624–934). GST pull-down was performed followed by an anti-His Western blotting analysis (upper panel). Protein expression of GST, GST-MSH2, GST-MSH2-(1–378), GST-MSH2-(200–700), and GST-MSH2-(624–934) was shown by Coomassie Blue staining (lower panel). E, diagrams of USP10 domain structure and USP10 deletion constructs. The numbers indicate the amino acids. UCH stands for the ubiquitin C-terminal hydrolase domain. F, C terminus of USP10 binds MSH2. Both wild type and catalytically-dead mutant of USP10 bind MSH2. Bacterially purified GST-MSH2 was incubated with 293T cell lysates transfected with Flag-empty vector (F-vector) or indicated USP10 constructs. The GST pull-down analyses were performed followed by an anti-Flag Western blotting analysis (upper panel). The input of Flag-USP10 deletion proteins and wild type (WT) and catalytically-dead mutant (C424A) of USP10 proteins was detected by an anti-Flag Western blotting analysis (middle panel). The amount of GST-MSH2 used for GST pull-down assays was examined by an anti-GST Western blotting analysis (lower panel).