(DOCX) Click here for additional data file.(14K, docx) Acknowledgments The authors thank Dr. were compared between GCNT1-positive and GCNT1-negative specimens. Survival was analyzed using Kaplan-Meier curves. Here, we raised a monoclonal antibody (mAb) against GCNT1 by immunization of a mouse with GCNT1 specific peptide (S1 File) to evaluate the potential of the latter as an indicator of PCa aggressiveness. In this study, we demonstrated that the anti-GCNT1 mAb showed high specificity against human GCNT1 and that GCNT1 expression in PCa specimens from radical prostatectomy correlates with PCa aggressiveness. In addition, detection of GCNT1 in post-digital rectal examination (DRE) urine by the anti-GCNT1 mAb predicted extracapsular extension of PCa. Therefore, detection of GCNT1 in post-DRE urine may serve as a minimally invasive method to predict PCa aggressiveness. Materials and Methods Materials ISOGEN II Reagent was purchased from Nippon Gene (Japan). A purified rabbit anti-mouse IgG antibody (-chain specific) was purchased from Zymed. A horseradish Galidesivir hydrochloride peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) antibody and an HRP-conjugated goat anti-mouse IgG antibody were acquired from Cell Signaling Technology. An HRP-conjugated goat anti-mouse IgG antibody was acquired from Millipore. Purified mouse myeloma protein from MOPC Mdk 21 (IgG, ), 2-mercaptoethanol, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. Tween-20 was purchased from Wako Pure Chemicals (Japan), as were DMEM and Hams F12 medium. Penicillin G/streptomycin solution was from Hyclone. Precision Plus Protein standards Dual Color were from Bio-Rad, and skim milk was from Yukijirushi (Japan). Cells Chinese hamster ovary (CHO) cells were Galidesivir hydrochloride maintained in the alpha modification of Eagle’s minimum essential medium Galidesivir hydrochloride (-MEM) supplemented with 100 U/mL of penicillin, 100 g/mL Galidesivir hydrochloride of streptomycin, and 10% fetal bovine serum (FBS). Immunohistochemical analysis of PCa specimens Between 2005 and 2011, 250 PCa patients were treated with radical prostatectomy at the Department of Urology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan. The tumor specimens were formalin-fixed and embedded in paraffin. Deparaffinized specimens were incubated with 5 g/mL of mouse anti-human GCNT1 mAb (clone HU127), Galidesivir hydrochloride followed by incubation with HRP-conjugated goat anti-mouse IgG antibody (H+L; Millipore). Immunoblotting analysis of post-DRE urine specimens Post-DRE urine was filled into 50 mL conical tubes, frozen immediately, and stored at -80C until analysis. Post-DRE urine specimens were collected from 35 patients who underwent radical prostatectomy from 2010 to 2013 at the Department of Urology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan. Frozen samples were thawed overnight at 4C and briefly centrifuged (5000 x em g /em , 5 min) to separate the supernatant and solids. Fifty microliters of the supernatant were spotted on to a nitrocellulose membrane. The membrane fixed with post-DRE urine protein was incubated with an anti-GCNT1 mAb (HU127), followed by an HRP-conjugated secondary antibody. Signals representing GCNT1 were enzymatically detected using the Novex? ECL Chemiluminescent Substrate Reagent Kit (Life Technologies) and visualized in a ChemiDocXRS+ System (Bio-Rad). Signal mean values were measured by Image Lab software (Bio-Rad). Amount of GCNT1 expression was calculated based on the transmission mean ideals of recombinant human being GCNT1 (R&D systems, 7248-GT). Auto-chemiliminescent signals were subtracted from total signals. Total protein concentration of post-DRE urine samples were measured by a BCA Protein Assay Kit (Pierce). Informed consent was from all individuals. All individuals offered their written educated consent to participate in this study. The honest committee of Hirosaki University or college approved the protocol of this study (The study about carbohydrate structure switch in urological disease; Authorization quantity: 2014C195). The study was performed in accordance with the honest requirements of the Declaration of Helsinki. Staging and grading of tumors All individuals were preoperatively evaluated using DRE, serum PSA screening, bone scanning,.