Fig. 4.

The SIM domain in E3 is required for its interaction with SUMO, its stability, SUMOylation, and nuclear localization, but it is not essential to bind dsRNA. (A) HEK-293 cells were transfected with WT-E3 or E3-ΔSIM expression plasmids and treated or not with MG132 for 24 h. Analyses of the protein extracts by Western blotting with an anti-E3 antibody were then carried out. (B) Protein extracts from HEK-293 cells transfected with WT-E3 or E3-ΔSIM expression constructs and treated with MG132 for 24 h were incubated with GST or GST-SUMO1. Beads were washed, and the bound proteins were eluted and subjected to electrophoresis and Western blot analysis with an anti-E3 antibody. The input, representing 10% of the protein extracts, is shown. (C) [³⁵S]methionine-labeled WT-E3 or E3-ΔSIM proteins were used as substrates in an in vitro SUMOylation assay in the presence of SUMO1 (top) or SUMO2 (bottom). The reaction products were visualized by autoradiography. (D) Total extracts or histidine-tagged purified proteins were prepared from HEK-293 cells cotransfected with pCINEO-E3-WT or pCINEO-E3-ΔSIM together with pcDNA or pcDNA-Ubc9 and pcDNA-His6-SUMO2, and an immunoblot analysis with an anti-E3 antibody was carried out. (E) MCF-7 cells were transfected with WT-E3 or E3-ΔSIM expression plasmids and treated with MG132 for 24 h. Cells were then stained with DAPI and an anti-E3 antibody, and the localization of E3 was detected by confocal microscopy. (F) [³⁵S]methionine-labeled WT-E3 or E3-ΔSIM proteins were incubated with agarose or poly(I:C)-agarose. Beads were washed, and the bound proteins were eluted and subjected to electrophoresis and autoradiography. The input, representing 10% of the quantity of protein used for the binding reaction, is shown.