Figure 5.

USP2 hydrolyses Imd-linked Ub^K48. A. Drosophila S2 cells were cotransfected with Imd-V5 and either wild type USP2 or mutant USP2C540S (USP2C*) expressing constructs in untreated cells or cells treated with MG132 at 20 μM for 4 hours before cell lysis. Imd-V5 was immunoprecipitated with anti-V5 antibodies (IP V5) and Imd-ubiquitinated forms were detected by western blot with either anti-Ub^K63 (WB K63) or anti-Ub^K48 (WB K48) antibodies as indicated. Imd full length was detected with anti-V5 antibodies (IP V5 WB V5). Expression of USP2 was assessed by western blot of whole cell lysate with anti-Myc antibodies (WB Myc). B. Drosophila S2 cells were cotransfected with Imd-V5 in the presence or not of Usp2 silencing dsRNA (dsUsp2). Imd-V5 was immunoprecipitated with anti-V5 antibodies and Imd-ubiquitinated forms were detected by western blot with anti-Ub^K48 antibodies (IP V5 WB K48). Imd full length was detected in the cell lysate with anti-V5 antibodies (WB V5). C. Endogenous Imd was immunoprecipitated with anti-Imd antibodies from extracts of c564/+ control flies (Ctrl), or flies expressing either the Usp2 silencing transgene (Usp2-IR), or the wild type (USP2) or the catalytically inactive form (USP2C*) of USP2, under the control of the c564Gal4 driver line (IP Imd). Ubiquitinated forms of Imd were detected with either Ub^K63 (WB K63) or Ub^K48 (WB K48) antibodies as indicated. The amount of endogenous Imd proteins was detected in total fly extracts with Imd antibodies (WB Imd). A-C. Anti-tubulin antibodies served as loading control (WB Tub).