glycerol and observed with a Nikon E 600 fluores- cence microscope equipped with a digital camera. Western blot analysis Antibody specificity to recombinant prelamin A (unprocessed protein precursor) or mature lamin A was tested by Western blot analysis. Recombinant proteins previously separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes were reacted with serial dilutions of purified anti- bodies 1188-1 and 1188-2. To evaluate antibody labeling in human fibrob- lasts, cells were lysed in Ripa buffer containing 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1mM PMFS, 1 μM aprotinin, leupeptin, pepstatin. Cell lysates were diluted in Laemmli buffer, subjected to SDS-PAGE (7.5%) and transferred to nitrocellulose mem- brane. Membranes were saturated with 4% BSA and incubated with primary antibodies for 1 hour at room temperature. Both anti-prelamin A 1188-1 and anti-prelamin A 1188-2 were applied at 1:100 dilution. Anti-lamin A/C antibody (Santa Cruz Biotechnology, SC-7292,) was applied at 1:100 dilution; anti-βactin-antibody was used at 1:1000 dilution as a loading control. Secondary antibodies were applied for 20 minutes at room temperature. Immunoblotted bands were revealed by the Amersham ECL detection system (GE Healthcare). Results and Discussion The complex mechanism of prelamin A process- ing has been summarized in Figure 1A. Among the prelamin A forms reported in the scheme, the two antibodies developed in this study were expected to detect full-length non-farnesylated prelamin A (preLA-CSIM) and farnesylated prelamin A lack- ing the C-terminal SIM sequence (preLA-farnesyl- C), respectively. Our results clearly show that anti- body 1188-1 detects full-length non-farnesylated prelamin A (preLA-CSIM) and, with lower affinity, also full length farnesylated prelamin A (preLA- farnesyl-CSIM). Moreover, antibody 1188-2 specif- ically recognises farnesylated prelamin A lacking the C-terminal SIM sequence (preLA-farnesyl-C), but not the full-length protein. To our knowledge, antibody 1188-2 is the first antibody that selective- ly binds preLA-farnesyl-C or preLA-farnesyl-C- CH3. Original Paper Data obtained in human skin fibroblasts demon- strate that a huge amount of full-length non-farne- sylated prelamin A (preLA-CSIM) is accumulated in fibroblasts by inhibition of protein farnesylation, while a significant amount of full-length farnesylat- ed prelamin A is accumulated by blockade of ZMP- STE24 activity. Moreover, we show that car- boxymethylated farnesylated prelamin A (preLA- farnesyl-C-CH3), although clearly detectable in progeria cells as the truncated form called prog- erin, is recovered in low amounts when ZMPSTE24 activity is inhibited. Antibody 1188-1 selectively binds full-length prelamin A in vitro Antibody 1188-1 was raised against the last 18 aminoacids of the prelamin A sequence. Post-trans- lational modifications were not introduced in the peptide used to immunize rabbits, as reported above (Figure 1B). Antibody characterization was performed by ELISA on plates coated with the pep- tide LLGNSSPRTQSPQNCSIM or plates coated with recombinant full-length prelamin A (preLA- CSIM). 1188-1 antibody was also tested on plates coated with LLGNSSPRTQSPQNC-Farnesyl pep- tide (peptide 1188-2) and recombinant mature lamin A. The results clearly demonstrate that the antibody 1188-1 displays high affinity for the pep- tide LLGNSSPRTQSPQNCSIM (Figure 2A). A low cross reactivity of 1188-1 antibody on peptide 1188-2 was observed only at very high concentra- tions (Figure 2B). Moreover, antibody 1188-1 showed high affinity to full-length unprocessed prelamin A (preLA-CSIM), as determined on recombinant prelamin A-coating (Figure 2C), but did not bind recombinant mature lamin A (Figure 2D).The specificity of 1188-1 antibody to prelamin A was confirmed by Western blot analysis on recombinant proteins (not shown). Antibody 1188-2 selectively binds carboxymethy- lated farnesylated prelamin A in vitro Antibody 1188-2 was raised against a farnesylat- ed peptide made up of the last 15 aminoacids of prelamin A, lacking the terminal SIM sequence (preLA-farnesyl-C, Figure 1C). ELISA performed to check antibody specificity was done on plates coated with the synthetic peptide LLGNSSPRTQSPQNC-Farnesyl (peptide 1188-2). The same antibody solutions were tested on LLGNSSPRTQSPQNCSIM peptide (peptide 1188- 47