(Coffinier et al., 2008). Figure 3B shows that cells treated with AFCMe or the combination of lopinavir-ritonavir or atazanavir-ritonavir are posi- tive to 1188-1 staining, suggesting that even farne- sylated prelamin A can be recognized by the anti- body, provided that the SIM sequence has not been removed. The presence of enlarged nuclei in AFCMe-treated cells (Figure 3B) is consistent with accumulation of farnesylated prelamin A (Mattioli et al., 2008). On the other hand, intranuclear stain- ing might suggest that full-length non farnesylated prelamin A (preLA-CSIM) is also accumulated in HIV protease inhibitor-treated cells. Noteworthy, atazanavir and amprenavir did not induce nuclear staining with 1188-1 antibody, indicating, in accor- dance with our previous results (Mattioli et al., 2008), that they do not inhibit ZMPSTE24 (not shown). Antibody 1188-2 reacts with carboxymethylated- farnesylated prelamin A in human fibroblasts To check the specificity of antibody 1188-2 we again used FTI-277 or mevinolin to induce accu- mulation of non-farnesylated prelamin A in human fibroblasts. Moreover, we used AFCMe and HIV protease inhibitors to induce accumulation of far- nesylated prelamin A forms, as reported above. In addition, we reasoned that the only cell model shown to accumulate a carboxymethylated and far- nesylated prelamin A form is represented by HGPS cells (Dechat et al., 2007). In those cells prelamin A is accumulated as progerin, a truncated prelamin A form obtained by alternative splicing, which lacks the second ZMPSTE24 cleavage site (Eriksson et al., 2003).Therefore, we checked HGPS fibroblasts for antibody 1188-2 staining of progerin. The results reported in Figure 3A show that human control cells either untreated or accumulat- ing non-farnesylated prelamin A by FTI-277 or mevinolin treatment are negative to 1188-2 anti- body staining at the nuclear periphery, while a faint intranuclear labelling is detected (Figure 3A). This result shows that the 1188-2 antibody does not rec- ognize non-farnesylated prelamin A. On the other hand, in cells treated with AFCMe low staining by 1188-2 antibody was observed (Figure 3B), as well as in fibroblasts subjected to HIV protease inhibitor treatment (Figure 3B). The antibody labelled the nuclear rim, but also the nucleoplasm. The latter was an unexpected finding, which was however supported by experiments performed in Original Paper pathological models, using different antibodies (our unpublished results). Moreover, nucleoplasmic localization of prelamin A had been previously reported in a human cell line (Gruber et al., 2005). Data obtained in HGPS were striking. Since the antibody was obtained by taking advantage of the antigenicity properties of the farnesylated prelamin A C-terminus lacking the SIM sequence, we hypoth- esized that it had to be able to recognize progerin, the truncated prelamin A form accumulated in HGPS, which had been shown to harbour that sequence (Dechat et al., 2007). In fact, we show that the highest labelling efficiency of 1188-2 anti- body is observed in HGPS fibroblasts, showing a strong nuclear rim staining in highly dysmorphic nuclei and a nuclear rim and intranuclear staining in ovoid nuclei (Figure 3B). It should be noted that antibody 1188-2 still labelled progerin when applied at higher dilution (Figure 3B), while it failed to label AFCMe-treated cells at this dilution (not shown). These results demonstrate that anti- body 1188-2 selectively and efficiently labels car- boxymethylated farnesylated prelamin A. Western blot analysis of drug-treated fibroblasts shows high specificity of antibodies Figure 4 shows the Western blot analysis of prelamin A in different samples performed using 1188-1 or 1188-2 antibody. In lysates from untreated control fibroblasts, antibody 1188-1 failed to reveal any protein band (Figure 4). Lysates from FTI-277 or mevinolin-treated cells, which are expected to accumulate preLA-CSIM, were strongly positive for 1188-1 staining (Figure 4). Cellular lysates from AFCMe-treated fibrob- lasts were also positive for anti-prelamin A 1188-1 staining, but a reduced amount of protein was detected, despite equal protein loading (Figure 4). This could be due to a lower affinity of the antibody for the farnesylated form of the full-length prelamin A. However, a sharp prelamin A band was detected with 1188-1 in samples treated with indinavir, nel- finavir, or the combination of lopinavir-ritonavir or atazanavir-ritonavir, while no staining was detected in amprenavir or atazanavir-only treated cells (Figure 4). These findings suggest that AFCMe and HIV protease inhibitors could affect prelamin A processing to different extent. 1188-1 antibody did not label any protein band in HGPS cellular lysates, suggesting that a minimal amount of full- length prelamin A, if any, is accumulated in those 49