Figure 5
USP2 hydrolyses Imd-linked UbK48. 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-UbK63 (WB K63) or anti-UbK48 (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-UbK48 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 UbK63 (WB K63) or UbK48 (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).