S. Dominici et al. Figure 4. Prelamin A Western blot analysis in control and HGPS skin fibroblasts. Western blot analysis of human fibroblast lysates per- formed with antibody 1188-1, 1188-2 or anti-lamin A/C antibody is shown. (A) Analysis of cells forced to accumulate non-farnesylated prelamin A. Cellular lysates have been obtained from control untreated fibroblasts (control), or from fibroblasts treated with FTI-277 or mevinolin to accumulate non-farnesylated prelamin A. (B) Analysis of cells accumulating farnesylated prelamin A. Cells were treated with the HIV protease inhibitors atazanavir, amprenavir, indinavir, nelfinavir, ritonavir+lopinavir and ritonavir+atazanavir (left panels) to accumu- late farnesylated prelamin A. Analysis of lysates from AFCMe -treated control fibroblasts and untreated HGPS fibroblasts, known to accu- mulate farnesylated prelamin A, is shown in the right panels. Atazanavir and amprenavir did not affect prelamin A processing and are used as negative controls. Actin is labelled as a loading control. Asterisks indicate the shift of the prelamin A band detected by anti-lamin A/C antibody. cells (Figure 4). Lysates from untreated and mevi- nolin or FTI-277-treated cells were not labelled with 1188-2 antibody, showing that the antibody 1188-2 does not cross-react with mature lamin A or with preLA-CSIM (Figure 4). A faint 74 kDa prelamin A band was labelled by 1188-2 antibody in AFCMe-treated fibroblast lysates, and in cells treated with indinavir, nelfinavir, or combinations of HIV-protease inhibitors containing ritonavir (Figure 4). Importantly, a sharply labelled progerin band (68 kDa) was detected using 1188-2 antibody in HGPS cell lysates (Figure 4). Since only the last four aminoacids of the peptide used to produce 1188-2 and the farnesyl residue are present in the progerin sequence (Eriksson et al., 2003), we can conclude that such C-terminus sequence is suffi- cient for the binding of 1188-2 antibody. Thus, we can show the following reactivity of anti-prelamin A antibodies in human fibroblasts. Antibody 1188-1 binds full-length prelamin A both in its non-farnesylated and in its farnesylated form (preLA-CSIM and preLA-farnesyl-CSIM). The far- nesylated form of full-length prelamin A appears to be labelled by the antibody with lower efficiency. Antibody 1188-2 efficiently binds farnesylated 50 prelamin A devoid of the SIM C-terminal sequence, following the first endoprotease cleavage, while it does not recognise full-length prelamin A. Further studies are needed to discriminate whether or not antibody 1188-2 binding is affected by prelamin A carboxymethylation. Inhibition of ZMPSTE24 causes accumulation of full-length farnesylated prelamin A Data previously reported show that AFCMe and HIV protease inhibitors used in this study impair ZMPSTE24 activity and cause accumulation of farnesylated prelamin A (Kilic et al., 1997; Caron et al., 2007; Coffinier et al., 2007; Coffinier et al., 2008; Mattioli et al., 2008). In our experimental models, carboxymethylated farnesylated prelamin A is detectable at very low levels after treatment with endoprotease inhibitors. On the other hand, a significant amount of full-length prelamin A is accumulated, as indicated by the strong reactivity of antibody 1188-1 in AFCMe- or HIV protease inhibitor-treated cells.Therefore, our results strong- ly suggest that blocking ZMPSTE24 activity in human cells, mostly impairs the first cleavage step of prelamin A (step 2 in Figure 1A) and only a