Supplementary Material for: The Search for Secreted Proteins in Prostate Cancer by the <b><i>Escherichia coli</i></b> Ampicillin Secretion Trap: Expression of NBL1 Is Highly Restricted to the Prostate and Is Related to Cancer Progression

<b><i>Aims:</i></b> Genes expressed only in cancer tissue or specific organs will be useful molecular markers. To identify genes that encode secreted proteins present in prostate cancer (PCa), we generated <i>Escherichia coli</i> ampicillin secretion trap (CAST) libraries from PCa and normal prostate (NP). <b><i>Methods and Results:</i></b> We identified 15 candidate genes that encode secreted proteins present in PCa and NP. Quantitative RT-PCR analysis revealed that <i>MSMB</i>, <i>NBL1</i> and <i>AZGP1</i> were expressed with much higher specificity in PCa and NP than in 14 other kinds of normal tissue. We focused on <i>NBL1</i>, which was originally identified as a putative tumor suppressor gene. Western blot analysis revealed that NBL1 protein was highly expressed in both cell lysate and culture media of the DU145 PCa cell line. Immunohistochemical analysis showed that NBL1 expression was highly detected in and restricted to NP and PCa and was significantly down-regulated in PCa. NBL1 expression was significantly reduced according to the tumor stage, Gleason grade and preoperative prostate-specific antigen (PSA) value. <b><i>Conclusion:</i></b> NBL1 is a secreted protein that is highly restricted to the prostate. Underexpression of NBL1 correlated with PCa progression. NBL1 might be a candidate tumor marker for PCa in addition to PSA.