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Figure 1 | Retrovirology

Figure 1

From: MDM2 is a novel E3 ligase for HIV-1 Vif

Figure 1

MDM2 downregulated cellular Vif levels in a proteasome dependent manner. (A) MDM2 reduced cellular levels of Vif as well as p53, but not that of Nef. HEK293T cells were cotransfected with expression vectors for the indicated E3 ligases and a subgenomic HIV-1 expression vector pNL-A1. Cell lysates were subjected to immunoblotting with the indicated Abs. We could not detect the expression of FLAG-MDM2 without MG132 treatment, because of a rapid degradation of MDM2. MG132 treatment enabled us to detect expression of MDM2 only with anti-MDM2 Ab, but not with anti-FLAG mAb. (B) Twenty-two hours after transfection, the cells were treated with cycloheximide (CHX)(80 μg/ml) for the indicated times, and cell lysates were subjected to immunoblotting with the indicated Abs. (C) The amounts of Vif and Nef were quantified by densitometry, and Vif protein levels were calculated using Nef protein levels as normalizing loading controls and presented as percentage values relative to that without CHX treatment set as 100%. Values are presented as averages of three independent experiments. (D) MDM2 downregulated Vif, but a ΔRF mutant did not. HEK293T cells were cotransfected with expression vectors for MDM2 and the mutant together with pNL-A1, and cell lysates were subjected to immunoblotting with the indicated Abs. (E) p53-/-MDM2-/- DKO-MEF cells were cotransfected with expression vectors for MDM2 and Vif, and treated with 10 μM MG132 for 6 hrs, and cell lysates were subjected to immunoblotting with the indicated Abs.

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