Figure 2From: Inhibition of HIV-1 gene expression by Ciclopirox and Deferiprone, drugs that prevent hypusination of eukaryotic initiation factor 5ACiclopirox 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.Back to article page