Figure 5.

Tak1 is required for Imd phosphorylation and ubiquitin editing. A–C, requirement of Tak1 for Imd phosphorylation and ubiquitin editing. S2* cells were treated with dsRNA (A and B) or the Tak1 inhibitor (5Z)-7-oxozeaenol (C), followed by PGN stimulation for indicated times. Modifications of endogenous Imd were assayed as previously described. Tak1 RNAi efficiency was monitored by qRT-PCR (B). Data are shown as mean ± S.D. from three independent experiments. IP, immunoprecipitated; IB, immunoblotting. D, requirement of Tak1 in Imd phosphorylation in vivo. Control (yw;wt) and yw;Tak1² adult flies were challenged with live E. coli by septic infection for indicated times. Endogenous Imd was assayed as above. E, Tak1 expression is sufficient to drive Imd phosphorylation. Stable cell lines expressing FLAG-Tak1 from the copper-inducible metallothionein promoter were treated with CuSO₄ (500 μm) for 6 h followed by PGN stimulation, as indicated. Phosphorylation of endogenous Imd was assayed by immunoprecipitated and immunoblotting. Tak1 protein level was monitored by lysate immunoblotted against FLAG. All blots are representative of at least three independent experiments. In all cases, the open triangle marks unmodified full-length Imd, the open arrow marks cleaved Imd, and the black arrows mark phosphorylated Imd.