Figure 4

USP2 interacts with Imd. A. Representation of the constructs used in this study. DD: death domain in Imd, CD: catalytic domain in USP2, V5: V5 tag, Myc: Myc tag. Numbers in brackets indicate amino-acid position. B. Drosophila S2 cells were cotransfected with the expression constructs encoding USP2-Myc and either Imd-V5, or Imd-N- ter-V5 or Imd-C- ter-V5 as indicated. Cells lysates were coimmunoprecipitated (IP) with either anti-V5 or anti-Myc antibodies and analysed by western blot (WB) with either anti-Myc or anti-V5 antibodies as indicated. C. S2 cells were transfected with the expression construct encoding Imd-V5 and lysed after 48 h. Cells lysates were pre-cleared and subjected to GST pull down assays using GST fusion proteins with either the USP2-N-ter [1–531] or the USP2-C-ter [475–856] - or with GST alone (GST). Gel was coloured with Coomassie to visualize GST-fusion proteins in the input (bottom part). D. GST-fusion of the wild type (USP2CD) or mutated (USP2CDC540S indicated USP2CD*) catalytic domain of USP2 were coexpressed with Ub-β-gal for 4 hours at 28°C in transformed E. coli XL1 Blue. Substrate cleavage was analysed by western blotting with anti-βgal antibodies.