FIG. 2.

The N-terminal domain of EBNA3C binds to Mdm2. (A) Fifteen million HEK 293T cells were cotransfected with pRK5-HA-Mdm2, encoding Mdm2 tagged with the HA epitope, or pA3F-EBNA3C proteins, encoding either full-length EBNA3C (1 to 992) or different truncated versions (residues 1 to 365, 366 to 620, and 621 to 992), as indicated. All EBNA3C proteins were tagged with Flag epitope. Cells were harvested at 36 h, 5% of the lysed cells were saved as input, and the remainder were IP with 1.5 μg anti-Flag antibody (M2). Samples were resolved by 10% SDS-PAGE and transferred to an 0.45-μm nitrocellulose membrane. The membrane was probed with anti-HA antibody (12CA5) followed by an infrared-tagged secondary antibody and scanned. The membrane was stripped and reprobed with M2 antibody followed by an infrared-tagged secondary antibody and rescanned. (B to D) Full-length or different truncated mutant constructs of EBNA3C (B and C) and Mdm2 protein (D) were in vitro translated (IVT) using a T7-TNT translation kit. All ³⁵S-radiolabeled in vitro-translated proteins in binding buffer were precleared by rotation with GST beads for 1 h at 4°C unless otherwise stated. Binding reactions were set up by incubating the in vitro-translated proteins with either GST control or GST fusion proteins (GST-Mdm2 [B and C] and GST-EBNA3C 90 to 130, GST-EBNA3C 130 to 160, and GST-EBNA3C 160 to 190 [D]). Reaction mixtures were resolved on appropriate polyacrylamide gels, exposed to phosphorimager plates, and scanned on a Storm 850 imaging system. Coomassie blue staining of SDS-PAGE-resolved purified GST-EBNA3C truncated proteins is shown at the bottom of panel D. All panels are representative gels from similar repeated experiments. (E) The schematic illustrates structural motifs of EBNA3C that potentially contribute to protein-protein interactions and summarizes the study of binding between different domains of EBNA3C and full-length Mdm2. ++, strong binding; +, moderate binding; −, no binding. NLS, nuclear localization signal. Numbers at left of gels and blots are molecular masses in kilodaltons. The asterisk indicates the immunoglobulin bands.