FIG. 4.

Direct interactions between IE2-86 and mdm2. (A) In vitro translation reveals direct interaction between IE2-86 and mdm2. GST-tagged IE2-86 and its deletion mutants were expressed in bacteria (as verified by Western blotting; data not shown) and collected on glutathione-Sepharose beads that were incubated with [³⁵S]mdm2 produced by in vitro transcription-translation. Specifically bound mdm2 was determined by autoradiography. Complexes formed by [³⁵S]mdm2 and GST-IE2-86 or its deletion mutants were analyzed by SDS-PAGE and autoradiography (upper panel) and expression of GST-IE2 and its deletion mutants in BL21 cells was determined by SDS-PAGE and Coomassie blue staining (bottom panel). (B) Coimmunoprecipitation of IE2-86 and mdm2 from lysates of MG132-treated or HCMV-infected HEL cells. The upper panel shows Western blots for mdm2 and IE2-86 in cell lysates. HEL cells were treated with 20 μM MG132 for 10 h prior to and 8 h postinfection (lanes 2 and 3) or 30 μM MG132 for 10 h (lane 5) after the time at which the cells in lanes 3, 4, and 6 were infected. The bottom panel shows immunoprecipitation with anti-IE2 antibody and Western blots for mdm2 and IE2-86. Lanes 1 to 4 received the same treatments in the upper and lower panels. In lane 5+6, the lysates of lanes 5 and 6 from the upper panel were mixed at 4°C for 1 h and the mixture was subjected to immunoprecipitation with subsequent Western blotting. IP, immunoprecipitation; W, Western blotting; Ub, ubiquitin; IgG, immunoglobulin G.