Figure 4

MDM2 induced the polyubiquitination of Vif in vitro and in vivo. (A) GST-MDM2 induced the polyubiquitination of Vif in vitro. Bacterially expressed GST-Vif was subjected to in vitro ubiquitination assays. The reaction was performed in the presence or absence of E1, E2, GST-MDM2, and GST-Ubiquitin as indicated. Reactions were subjected to immunoblotting with anti-Vif mAb. Arrows indicate GST-ubiquitin-conjugated Vif. (B) Overexpressed MDM2 induced the polyubiquitination of Vif in vivo. HEK293T cells were cotransfected with expression vectors for MDM2 Wt and a ΔRF mutant together with expression vectors for Vif and His-Ubiquitin (His-Ub) as indicated. Cells were treated with MG132 for 6 hrs, and cell lysates were precipitated with Ni-NTA agarose beads followed by immunoblotting with the indicated Abs. Since Vif naturally bound to Ni-NTA agarose, we detected a Vif band itself (arrowhead), whereas no signal was detected in cells lacking Vif (lane 3). Arrows indicate His-Ub-conjugated Vif. Arrows with asterisk indicate Vif conjugated with endogenous ubiquitin. (C) Transduction of siRNA reduced cellular levels of endogenous MDM2 and polyubiquitination of Vif. HEK293T cells were cotransfected with expression vectors for MDM2 siRNA and control siRNA together with expression vectors for Vif and HA-Ubiquitin (HA-Ub). Cell lysates were immunoprecipitated with anti-Vif mAb followed by immunoblotting with the indicated Abs. Asterisk indicates immunoglobulin light chains from the immunoprecipitation.