0) and immobilized on the carboxymethyl dextran surface of the cuvette, according to the manufacturer’s instructions. Binding experiments were performed in PBS. Changes in the resonant angle were monitored at 1-s intervals for approximately 600 s. Experiments were performed at 25��C normally with a stirrer speed of 80 rpm. The binding parameters were calculated from the association and dissociation phases of the binding reactions using the non-linear curve fitting FastFit (Affinity Sensors). Bovine serum albumin (BSA) was used as a control. Microarray data The clinical samples of the paired colorectal cancers (CRCs), microarray procedure and analysis method have been previously described [7]. This study was approved by the institutional review board, and written informed consent was obtained from all the patients.

All microarray data has been deposited to Center for Information Biology gene Expression database (CIBEX, http://cibex.nig.ac.jp/index.jsp) as accession number #CBX205. All data is MIAME compliant and that the raw data has been deposited in a MIAME compliant database (CIBEX), as detailed on the MGED Society website http://www.mged.org/Workgroups/MIAME/miame.html. Patients and samples The 30 CRC and 10 paired non-cancerous colonic mucosa samples were analyzed using real-time RT-PCR. The RNA extraction method and the quality check protocol have been previously described [7]. This study was approved by the institutional review board of the National Cancer Center Hospital, and written informed consent was obtained from all the patients.

Real-time reverse transcription PCR and western blot analysis The methods used in this section have been previously described [5]. Results Overexpression of SRPX2 in CRC tissues We evaluated the mRNA expression of SRPX2 in clinical samples of CRCs in addition to its homologue SRPX (SRPX1) using microarray data. SRPX2 expression was markedly up-regulated (20.5 fold, p=0.00014) in cancer tissues, compared with paired noncancerous mucosa samples, whereas the putative tumor suppressor gene SRPX was down-regulated (0.7 fold, p=0.029) in cancer (Fig. 1). The result indicates that SRPX2 is overexpressed in CRC during carcinogenesis and tumor progression, unlike SRPX. Real-time RT-PCR for the 30 CRC and 10 paired non-cancerous colonic mucosa samples confirmed that SRPX2 mRNA was markedly overexpressed in the CRC samples but was only expressed at a very low level in non-cancerous colonic mucosa (Figure 1B).

Figure 1 SRPX2 is overexpressed in colorectal cancer (CRC). Secreted SRPX2 protein is suspected to be modified posttranslationally The predicted molecular mass of SRPX2 protein was 53 kDa; however, western blotting revealed that the molecular mass of the secreted SRPX2 protein was highly increased, with smeared Batimastat bands at an apparent molecular mass of 100�C150 kDa in SNU-16 and MKN7 cell lines (Fig. 2A). Next, we evaluated the exogenously expressed SRPX2 protein derived from HEK293-Mock and HEK293-SRPX2-HA/His cells.