FIG. 3.

Mdm2 interacts with 16E2 in vitro and in vivo. (a) In vitro-translated and radiolabeled Mdm2 was incubated with bacterially purified GST-tagged E2. GST alone and GST-p53 were included as negative and positive controls, respectively. Bound proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The Coomassie blue stains of the GST inputs are also included in the top panel. An asterisk indicates the full-length GST fusion proteins. (b) Mdm2 binds to the C-terminal region of E2. GST-16E2 and a number of GST-tagged fragments of E2 (right) were incubated with in vitro-translated and radiolabeled Mdm2, and bound proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Fifty percent of the input is included, and the GST inputs are shown in the bottom panel and are stained with Coomassie blue. An asterisk indicates the full-length GSTs. NE2, N-terminal half of E2 protein; CE2, C-terminal half of E2 protein. (c) E2 and Mdm2 bind in vivo. 293 cells were transfected using either Mdm2 alone or Mdm2 with GFP-tagged E2. Cell extracts were immunoprecipitated (IP) using polyclonal anti-GFP antibodies, followed by Western blot analysis using antibodies against Mdm2 or GFP. An asterisk indicates nonspecific bands. -ve, untransfected cells.