S. Dominici et al. 1) or recombinant full-length prelamin A-coated plates. The results demonstrate that antibody 1188-2 reacts with 1188-2 peptide, but not with 1188-1 peptide (Figure 2A, B). Moreover, ELISA performed on full-length prelamin A (preLA- CSIM)-coated wells showed no cross-reactivity of the antibody (Figure 2C, D). The specificity of anti- body 1188-2 to prelamin A was further investigat- ed by Western blot analysis. As expected, no immunoreactive bands were detected on any of the two proteins (data not shown), showing that anti- body 1188-2 does not recognize full-length prelamin A or mature lamin A. Antibody 1188-1 reacts with non-farnesylated prelamin A in human fibroblasts Antibody 1188-1 was used to detect prelamin A forms in control human fibroblasts either untreated or treated in vivo with prelamin A processing inhibitors. Antibody 1188-1 did not stain the nuclear rim in untreated human fibroblasts (Figure 3A). A faint labelling of intranuclear aggregates was observed in a minor percentage (<10%) of cells (not shown). Following FTI-277 or mevinolin treatment, which cause accumulation of non-farne- sylated prelamin A (preLA-CSIM) (Verstraeten et al., 2006; Mattioli et al., 2008), human fibroblasts were highly positive to 1188-1 antibody staining, showing a clear nuclear rim labelling (Figure 3A). Moreover, intranuclear prelamin A aggregates were sharply labelled (Figure 3A), as expected for non- farnesylated prelamin A (Lattanzi et al., 2007; Mattioli et al., 2008). Antibody 1188-1 detects full-length farnesylated prelamin A in human fibroblasts Since we could not rule out the possibility that antibody 1188-1 might detect farnesylated full- length prelamin A (preLA-farnesyl-CSIM), we checked antibody reactivity in cells accumulating farnesylated prelamin A. In order to induce the accumulation of farnesylated forms of prelamin A, we used a set of drugs known to block ZMPSTE24 binding or activity. The non-peptidomimetic com- pound N-acetyl-S-farnesyl-L-cysteine methylester (AFCMe) impairs ZMPSTE24 binding to prelamin A (Corrigan et al., 2005; Mattioli et al., 2008), while the HIV protease inhibitors are known to cause prelamin A accumulation, and lopinavir has been recently shown to inhibit ZMPSTE24 activity and cause accumulation of farnesylated prelamin A 48 Figure 3. Prelamin A staining in control and HGPS skin fibrob- lasts. Prelamin A staining is shown in human control fibroblasts untreated (control) or treated with prelamin A processing inhibitors and in HGPS fibroblasts. (A) Fibroblasts untreated or treated with FTI-277 or mevinolin to accumulate non-farnesylat- ed prelamin A (preLA-CSIM). Antibody 1188-1 labeling is report- ed in the upper row, antibody 1188-2 labeling is reported in the lower row. (B) Fibroblasts untreated or treated with AFCMe or HIV protease inhibitors (combinations of lopinavir plus ritonavir and atazanavir plus ritonavir, as indicated) to accumulate farne- sylated prelamin A. HGPS fibroblasts, which also accumulate a farnesylated prelamin A form, are on the right. Antibody 1188-1 labeling is reported in the upper row, antibody 1188-2 labeling is reported in the lower row. (C) HGPS fibroblasts treated with mevi- nolin for 48 hours to show reduced staining using 1188-2 anti- body with respect to bright staining observed in untreated HGPS cells shown in (B) and (D). Prelamin A staining was revealed by FITC-conjugated anti-rabbit IgG (green). Bars, 10 μm.