Figure 6
Imd proteasomal degradation requires USP2. A. S2 cells were transfected by Imd-V5 expressing construct and treated or not by Usp2 silencing dsRNAs (dsUsp2). Cells were incubated or not with MG132 at 20 μM. Cell lysates were immunoblotted with anti-V5 antibodies (WB V5). B. Protein extracts of c564/+ control or c564/Usp2-IR flies were immunoblotted with anti-Imd antibodies (WB Imd). C. S2 cells lysates treated or not with dsUsp2 were immunoblotted with anti-Cact antibodies (DSHB) (WB Cact). D. Endogenous Imd was immunoprecipitated from extracts of c564/+ or c564/Usp2-IR flies infected or not by E. coli. Ubiquitinated forms of Imd were detected with anti-UbK48 antibodies (IP Imd WB K48). Total extracts were immunoblotted with anti-Imd antibodies (WB Imd). E. Endogenous Imd was immunoprecipitated from extracts of c564/+ or c564/Pros26[[1]]; Prosbeta[[1]]/+ flies (c564 > ProsDN). Ubiquitinated forms of Imd were detected by anti-UbK48 antibodies (IP Imd WB K48). A-E: Anti-tubulin immunoblots served as loading control (WB Tub). F. Quantitative analysis of Dpt and AttA mRNAs from c564 > ProsDN compared to c564/+ control flies. Histograms present the fold activation of each mRNA. Error bars indicate standard deviation between technical triplicates. Significant differences with control (t-test) p < 0.001 (***). G. S2 cells were cotransfected with pAc-USP2-Myc and either pAc-Imd or a control empty plasmid (Ctrl). USP2 was immunoprecipitated with anti-Myc antibodies and immunoblotted with anti Pros45 antibody (DHSB) (IP Myc WB Pros45). Cells lysates were immunoblotted with anti-Myc and anti Pros45 antibodies (WB Myc, WB Pros45). H. S2 cells were cotransfected with pAc-Imd-V5 and either pAc-Usp2 or a control empty plasmid (Ctrl). Imd was immunoprecipitated with anti-V5 antibodies and endogenous Pros45 was detected (IP V5 WB Pros45). Whole cells lysates were revealed with anti-V5 and anti Pros45 antibodies (WB V5 WB Pros45).