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. 2007 Jul 25;104(33):13432–13437. doi: 10.1073/pnas.0704212104

Fig. 3.

Fig. 3.

HIV-PIs block the processing of an enhanced GFP-prelamin A fusion protein. (A) Schematic representations of the GFP-prelamin A fusion protein and its cleavage by ZMPSTE24. The cDNA sequences encoding amino acids 548–665 from mouse prelamin A (represented by the blue oval) were ligated in-frame to GFP (represented as a green oval). The location of the prelamin A cleavage reaction carried out exclusively by ZMPSTE24 (cleavage P2) is indicated, as are the locations of epitopes for antibodies against GFP, mature lamin A, and prelamin A. (B) Western blot analysis of HEK293 cells transiently transfected with a GFP expression vector (pEGFP-C1) or the GFP-prelamin A fusion construct. Antibody binding was detected with an Odyssey system (Li-Cor Biosciences). (Left) Red signal indicates binding of the anti-GFP antibody, and green signal indicates binding of the anti-mature lamin A antibody. The merged signals result in a yellow/green color. (Right) Red signal indicates binding of the anti-GFP antibody, and green signal indicates binding of the anti-prelamin A antibody. (C) Western blot, using an antibody against GFP, of WT and Zmpste24−/− fibroblasts transiently transfected with the GFP-prelamin A fusion construct and treated overnight with the vehicle (DMSO), LPV (20 μM), or the FTI (5 μM). (D) Western blot analysis of HeLa cells transiently transfected with the GFP-prelamin A fusion construct and treated overnight with vehicle (DMSO), LPV (20 μM), atazanavir (ATV) (20 μM), STV (20 μM), NVP (20 μM), or an FTI (5 μM). Western blot was performed with antibodies against GFP and prelamin A (Middle and Bottom). Top shows the merged image; the anti-GFP signal is red, and the anti-prelamin A signal is green. (E) Western blots of HEK293 cells transiently transfected with the GFP-prelamin A construct and treated overnight with LPV (20 μM), the FTI (5 μM), or vehicle (DMSO). (F) Western blots of HEK293 cells transiently transfected with the GFP-prelamin A construct and treated overnight with LPV (20 μM), TPV (20–100 μM), or DMSO.