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Figure 2 | Retrovirology

Figure 2

From: Inhibition of HIV-1 gene expression by Ciclopirox and Deferiprone, drugs that prevent hypusination of eukaryotic initiation factor 5A

Figure 2

Ciclopirox and deferiprone prevent the maturation of eIF5A. A. Drug inhibition of eIF5A modification in 293T cells. Cells transfected with FLAG-tagged eIF5A were untreated or treated with increasing concentrations of CPX as indicated, or with agent P2. At 24 hr post-transfection, whole cell extract (WCE) was analyzed by immunoblotting with the NIH-353 anti-eIF5A antibody (upper panel) and anti-actin antibody (lower panel). B. Cells transfected with FLAG-tagged eIF5A were untreated or treated with increasing concentrations of DEF as indicated, or with DFOX. Cells were processed as in A. C. Cells transfected with FLAG-tagged eIF5A were treated with CPX (30 μM), P2 (30 μM), DEF (250 μM), DFOX (10 μM), or no drug (-). At 24 hr post-transfection, WCE was analyzed by immunoblotting with the NIH-353 anti-eIF5A antibody (upper panel) and anti-FLAG antibody (lower panel). The control culture was transfected with empty vector and no drug was added. D. Inhibition of enzyme-substrate binding. 293T cells transfected with FLAG-eIF5A were untreated (-) or treated with GC7 (10 μM) or CPX (30 μM), P2 (30 μM), DEF (250 μM), or DFOX (10 μM). WCE prepared at 24 hr post-transfection was immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were immunoblotted with antibodies against DOHH (top panel) and FLAG (bottom panel). (*)-IgG light chain. E. 293T cells transfected with FLAG-DHS, FLAG-DOHH or empty vector (Control) were treated with GC7, CPX, or DEF, or no drug (-) at the same concentration as in panel D. Immunoprecipitates obtained with anti-FLAG antibody were immunoblotted and probed with anti-eIF5A antibody (BD). Input: WCE equivalent to 5% of the input was immunoblotted as a further control.

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