The amino acid sequence and the immunochemical characterization of the N- and C-terminal regions of RLIP76. (A) The nucleotide sequence of the cloned cDNA (GenBank accession number NM 006788) was divided in 2 subunits by PCR with specific primers. The cDNA subunits were cloned in an expression vector and the N- and C-terminal regions of the protein were expressed and purified. The overlapped amino acids were squared. (B) The molecular size and the purity of the expressed proteins were confirmed by 10% SDS-PAGE stained by Coomassie blue (lane 1, N-terminal region; lane 2, C-terminal region) and serum immunoreactivity was analyzed by immunoblotting (lanes 3-6, C-terminal region; lanes 7-10, N-terminal region). Lanes 3,7: monoclonal antibody specific to 6-histidine tail; lanes 4,8: serum pool from the 2 patients with Behçet disease (BD) used in screening the library; lane 5,9: representative serum from a healthy subject; lanes 6,10: control without serum.