S. Dominici et al. A post-translational modifications are lost in the mature protein (Rusinol and Sinensky, 2006), so that the complex mechanism leading to prelamin A maturation must play per se some functional role, which has not been completely unravelled. Data so far reported show that the lamin A precursor affects to some extent nuclear envelope targeting of the mature protein (Hennekes and Nigg, 1994), transcription factor translocation (Caron et al., 2003; Capanni et al., 2005) and chromatin organ- ization (Lattanzi et al., 2007; Mattioli et al., 2008). Moreover, an accumulation of mutated or wild-type prelamin A has been shown in several laminopathies, linked to LMNA or ZMPSTE24 gene mutations, including Hutchinson-Gilford prog- eria syndrome (HGPS), Restrictive Dermopathy (RD), Mandibuloacral dysplasia type A (MADA) and B (MADB), atypical Werner syndrome (WS), familial partial lipodystrophies and metabolic laminopathies (De Sandre-Giovannoli et al., 2003; Eriksson et al., 2003; Navarro et al., 2004; Capanni et al., 2005; Filesi et al., 2005; Young et al., 2005; Caron et al., 2007; Maraldi and Lattanzi, 2007; Maraldi et al., 2006, 2007). In addition, we and others have shown that some HIV protease inhibitors used in anti-retroviral therapy inhibit ZMPSTE24, leading to a secondary laminopathy (Caron et al., 2003; Caron et al., 2007; Coffinier et al., 2007; Coffinier et al., 2008). A number of published data show that prelamin A accumulation is toxic to cells and that protein toxi- city could be not only dependent on protein amount, but also on the post-translational modification har- boured by the precursor itself (Caron et al., 2007; Lattanzi et al., 2007b; Yang et al., 2008). Therefore, the availability of antibodies directed to different prelamin A forms could allow a relevant advance in the study of prelamin A effects. Here we report construction and validation of two different antibodies raised in rabbit against prelamin A C-terminus peptides. The first one, called 1188-1, has been developed against the last 18 aminoacids in the prelamin A C-terminus, including the CSIM motif. The second antibody, called 1188-2, has been obtained using a farnesy- lated prelamin A C-terminus peptide lacking the last three aminoacids in the CSIM sequence. Here we demonstrate that the 1188-1 antibody is able to bind non-farnesylated prelamin A and, with lower affinity, farnesylated full-length prelamin A. The second antibody, on the contrary, does not recognize 44 preLA-CSIM and selectively binds carboxylated or carboxymethylated prelamin A harbouring the far- nesyl residue. These antibodies could represent a useful tool to investigate the biological significance of prelamin A processing and to discriminate which prelamin A forms are accumulated in laminopathies (Maraldi and Lattanzi, 2007). Moreover, they can be used to monitor the effects of drugs acting on prelamin A processing, which are potential therapeutic tools in laminopathies featuring prelamin A accumulation (Caron et al., 2007) and in cancer therapy (Adjei et al., 2000; Ramirez et al., 2007). Using these antibodies in experimental models of prelamin A accumulation and in HGPS cells, we could determine that the first cleavage step of prelamin A processing is mostly catalysed by the ZMPSTE24 endoprotease, whose inhibition causes accumulation of full- length farnesylated prelamin A. Materials and Methods Antibodies preparation Peptides LLGNSSPRTQSPQNCSIM (peptide 1188-1) and LLGNSSPRTQSPQNC-Farnesyl (peptide 1188-2), which represent aminoacidic sequences involved in the lamin A maturation path- way, were obtained by chemical synthesis (BIO- SYNTHESIS, Lewisville, USA) (for peptide analy- sis see Figures 1 and 2). Peptides were converted into haptens by combination with keyhole limpet hemocyanin (KLH) used as carrier, in order to increase their immunogenic properties. Synthetic peptides KLH-conjugated emulsified with Incomplete Freund’s adjuvant (IFA) were injected into New Zealand White rabbits for antibody pro- duction. Plasma was collected by centrifugation (2,800 rpm for 10 min). Immunoglobulin fractions were purified from rabbit antiserum by Protein A affinity chromatography (Sigma). Immunoglo- bulins were eluted with sodium citrate 0.1 M pH 3.0 and immediately neutralized with 1 M TRIS (100 μl/mL). Antibody characterization Antibody specificity was tested by ELISA. LLGNSSPRTQSPQNCSIM and LLGNSSPRTQS PQNC-Farnesyl synthetic peptides (5 μg/mL) dis- solved in 50 mM NaHCO3 pH 9.6 or prelamin A and lamin A recombinant proteins (1 μg/mL) dis-