LRRC32 contains an N-terminal signal peptide and a transmembrane region. a) Analysis of the amino acid sequence of LRRC32 by SignalP 3.0 software predicted a putative N-terminal cleavage site between alanines 17 and 18. Alternative potential cleavage sites are predicted and indicated as vertical solid lines on amino acids 19, 20, and 22. b) To address the actual cleavage site, N-terminal and C-terminal GFP-tagged constructs were designed as depicted and generated to facilitate further analysis of surface expression and cleavage. SP = signal peptide. TM = transmembrane region. GFP = green fluorescent protein. Arrow = putative cleavage site. c) Anti-GFP immunoblot analysis of total lysates from C-and N- terminal expressing clones revealed 99 kD (29 kD GFP + 70 kD LRRC32) and 31 kD (29 kD GFP + 2 kD LRRC32 signal peptide) fusion proteins respectively. Expected sizes of uncleaved fusion proteins, based upon predicted protein sequences, are shown in parentheses. The difference in protein size between the C- and N-terminal fusion proteins confirms a cleavage site in the N-terminus of the protein between alanines 17 and 18.