S. Dominici et al. solved in 50 mM NaHCO3 pH 9.6 were incubated at 4°C for 18 hours in 96 well plates. A detailed description of recombinant human prelamin A and lamin A purification will be reported elsewhere. Briefly, recombinant prelamin A (rPrelamin A) had been translated as a 74 kDa full-length precursor, while lamin A had been translated as a 70 kDa mature protein using a mutated LMNA construct. After discarding peptide and protein excess, the plates were blocked with PBS containing 0.05% (v/v) Tween 20 and 1% (w/v) BSA for 1 hour at 37°C. After washing, 100 μL of immune serum diluted in PBS containing 1% (w/v) BSA were added to each well and incubated at 37°C for 1 hour. Plates were washed and an HRP-conjugated anti-rabbit antibody (Bio-Rad Laboratories) was added and incubated for 1 hour at 37°C. The immune reaction was developed using 2,2’-azino- bis 3-ethylbenzthiazoline-6-sulfonic acid as sub- strate dissolved in a Colour buffer (50 mM of sodi- um citrate pH 3.0 with 1 μl/mL of H2O2). The absorbance at 405 nm was measured using a microplate reader (Bio-Rad Laboratories). Cell cultures Skin fibroblast cultures were obtained from skin biopsies of healthy patients (mean age 24) under- going orthopaedic surgery, following a written con- sent. HGPS fibroblast cell cultures were established from a skin biopsy of a 5 year old patient undergo- ing genetic analysis. The protocol had been approved by the local ethical committees. The c.1824C>T/p.G608G variation within the LMNA gene was identified by direct sequencing as previ- ously described (De Sandre-Giovannoli et al., 2003). Cell cultures were established and cultured in Dulbecco’s modified Eagle’s medium supple- mented with 20% fetal calf serum (FCS) and antibiotics.The experiments were performed at pas- sages 3 to 10. Drug treatments Ten μM FTI-277 (Calbiochem, Merck Biosciences) or 20 μM mevinolin (Sigma-Aldrich) were applied to control fibroblasts for 18 hours. These drugs impair prelamin A farnesylation through different mechanisms. FTI-277 inhibits farnesyltransferase activity through a pep- tidomimetic mechanism (Mattioli et al., 2008), mevinolin is a statin causing inhibition of hydrox- ymethyl-glutaryl-synthase implicated in the farne- 46 syl pathway (Verstraeten et al., 2006). Both com- pounds elicit accumulation of non-farnesylated prelamin A, as reported in several studies (Glynn and Glover, 2005; Young et al., 2006; Caron et al., 2007; Mattioli et al., 2008). 1-N-acetyl-S-farnesyl- L-cysteine methylester (AFCMe, ALEXIS Biochemicals) was applied at 10 μM concentration in culture medium for 18 hours. AFCMe is a non- peptidomimetic compound known to impair binding of the ZMPSTE24 endoprotease to prelamin A, thus affecting proteolytic steps and causing accu- mulation of farnesylated prelamin A. Protease inhibitors used in HIV anti-retroviral therapy were employed in our study to induce accumulation of farnesylated prelamin A. These drugs include the known prelamin A processing inhibitors nelfinavir, indinavir, lopinavir and ritonavir and two molecules recently shown to have modest or no impact on prelamin A maturation (atazanavir and ampre- navir) (Caron et al., 2007; Coffinier et al., 2008). Human control cultured fibroblasts were continu- ously incubated for 7 weeks with HIV protease inhibitors at concentration close to their Cmax (indinavir 10 μM, nelfinavir 5 μM, atazanavir 5 μM, amprenavir 10 μM, and the combinations lopinavir 10 μM plus ritonavir 2 μM or atazanavir 5 μM plus ritonavir 2 μM, frequently used for the treatment of patients). Indinavir was provided by Merck Sharp & Dohme Laboratories (Clermont- Ferrand, France), nelfinavir by Agouron Pharmaceuticals (San Diego, CA, USA), ampre- navir by Vertex Pharmaceuticals (Cambridge, MA, USA) and lopinavir and ritonavir by Abbott (Rungis, France). Atazanavir was provided by Dr. S Azoulay (CNRS UMR 6001, University Nice- Sophia Antipolis, France). Immunofluorescence microscopy Human fibroblasts grown on glass coverslips were fixed in methanol at -20°C for 7 minutes, washed in PBS and subjected to immunofluorescence label- ing. Samples were incubated with primary antibod- ies overnight at 4°C in PBS containing 4% BSA to saturate non-specific binding sites, and with sec- ondary antibodies for 1 hour at room temperature. Anti-prelamin A 1188-1 was applied at 1:100 dilu- tion and anti-prelamin A 1188-2 was applied at 1:10 dilution. HGPS fibroblasts were also stained with 1188-2 antibody at higher further dilutions up to 1:150. Nuclei were counterstained with DAPI. Slides were mounted with an anti-fade reagent in