Basel, Switzerland). The extracts were boiled in denaturing loading buffer and separated in pre-cast tris-acetate 5-8% SDS gradient gels (Invitrogen, Tåstrup, Denmark) with trisacetate running buffer (Invitrogen), employing pre-stained broad-range molecular weight markers (Bio-Rad, Herlev, Denmark) . The separated proteins were transferred to PVDF membranes by wet blotting in transfer buffer containing 20% isopropanol, 20 mM tris-acetate and 0.1% SDS, followed by blocking with 5% skimmed-milk powder in TBS-T buffer. Membranes were incubated with the human monoclonal antibodies or a monoclonal mouse anti-exon 2 antibody, followed by incubation with a secondary anti-human or anti-mouse AP-conjugated antibody (Sigma, MO, USA) and developed using a 5-bromo-4-chloro-3-indoyl phosphate and nitroblue tetrazolium solution (Sigma). Baculovirus-produced proteins and a pre-stained ProSieve protein marker (Cambrex) were run on pre-cast 4-12% SDS gradient gels (Invitrogen, Tåstrup, Denmark) with NuPage MOPS SDS running buffer (Invitrogen). Proteins were transferred to a nitrocellulose membrane by wet blotting using a buffer of 20% methanol, 25 mM Tris and 192 mM glycine. Following blocking in 5% skimmed-milk powder in TBS-T buffer, membranes were incubated for 1 h with either a 1:5000 dilution of horseradish peroxidase-conjugated loading control antibody anti-V5 (R960-25, Invitrogen) or a 1:1000 dilution of PAM8.1. The PAM8.1-probed membrane was further incubated with a 1:1000 dilution of a secondary anti-human IgG antibody (P0214, Dako Cytomation). Membranes were developed using 3-amino-9-ethyl-carbazole tablets dissolved in acetone, 50 mM sodium acetate and 30% H2O2.