MDM2 bound Vif in its central domain. (A) Immunoprecipitation assays revealed the interaction of MDM2 with Vif in vivo. HEK293T cells were cotransfected with expression vectors for MDM2 and Vif and treated with MG132 for 6 hrs prior to harvest. Cell lysates were immunoprecipitated with anti-MDM2 mAb followed by immunoblotting with the indicated Abs (upper two panels). Cell lysates were also subjected to immunoblotting with the indicated Abs (lower two panels). (B) The interaction domain of MDM2 with Vif. HEK293T cells were cotransfected with expression vectors for HA-tagged MDM2 wild type (Wt) and mutants together with pNL-A1, and cell lysates were immunoprecipitated with anti-HA mAb followed by immunoblotting with the indicated Abs. Asterisk indicates immunoglobulin heavy chains from thenimmunoprecipitation. (C) Schematics of MDM2 mutants binding to Vif are shown.