FIG. 2.

The coiled-coil domain-containing region of NS binds to the central acidic domain of MDM2. (A) MDM2 binds to the coiled-coil domain-containing region of NS in cells. H1299 cells were transfected with Flag-tagged full-length NS (wt) or its deletion mutants, together with the HA-MDM2 plasmid. Cell lysates were immunoprecipitated with the anti-Flag antibody, followed by IB using the indicated antibodies. The lysates were also loaded directly onto a sodium dodecyl sulfate (SDS) polyacrylamide gel for IB, using the anti-HA antibody (bottom panel). (B) MDM2 binds to NS in vitro. About 200 ng of purified GST alone, full-length GST-NS, or GST-NS deletion mutants immobilized on glutathione beads was incubated with 200 ng of His-MDM2 purified from bacteria. Bound MDM2 was detected by IB with anti-MDM2 antibodies. The GST-NS fusion proteins were visualized by Coomassie blue staining. (C) Schematic diagram of NS protein indicates the MDM2-binding domain (black bar). BD, basic domain; AD, acidic domain; CC, coiled-coil domain; G4 and G1, putative GTP binding motifs. (D) GTP binding activity of NS is not required for NS binding to MDM2. H1299 cells were transfected with HA-MDM2, together with Flag-NS, Flag-NS^G1dm, Flag-NS^ct, or the control Flag vectors. Cell lysates were immunoprecipitated with the anti-Flag antibody, followed by IB using the indicated antibodies. The lysates were also loaded directly onto an SDS gel for IB using the indicated antibodies (left panels). (E) NS binds to the central acidic domain of MDM2. H1299 cells were transfected with plasmids encoding V5-tagged MDM2 fragments, together with the indicated Flag-NS plasmid. Cell lysates were immunoprecipitated with an anti-V5 antibody, followed by IB using the indicated antibodies. The lysates were also loaded directly onto an SDS gel for IB using anti-Flag antibody (bottom panel). (F) Schematic diagram of MDM2 protein indicating the NS binding acidic domain (AD) (black bar). ZF, zinc finger domain; RF, ring finger domain.